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PubMed1995

Independent signaling of grp78 gene transcription and phosphorylation of eukaryotic initiator factor 2 alpha by the stressed endoplasmic reticulum.

Perturbation of endoplasmic reticular (ER) function signals increased expression of the gene encoding the ER resident chaperone Grp78/BiP and rapid suppression of translational initiation accompanied by phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 alpha phosphorylation and grp78 mRNA induction were measured in GH3 pituitary cells subjected to varied degrees of ER stress to ascertain whether activation of an eIF-2 alpha kinase is involved in both events. grp78 mRNA was induced at low concentrations of ionomycin and dithiothreitol that did not provoke eIF-2 alpha phosphorylation or inhibition of amino acid incorporation. Mobilization of the bulk of cell-associated Ca2+ and the induction of grp78 mRNA occurred at comparable low concentrations of ionomycin, whereas phosphorylation of eIF-2 alpha and inhibition of protein synthesis required higher ionophore concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2 alpha phosphorylation in response to either stressor. Prolonged (17 h) cycloheximide blockade increased eIF-2 alpha phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was induced and eIF-2 alpha was dephosphorylated. Translational tolerance to ionomycin or dithiothreitol, accompanied by dephosphorylation of eIF-2 alpha, was observed whenever grp78 mRNA was induced. Induction of grp78 mRNA preceded significant eIF-2 alpha phosphorylation during treatment with brefeldin A. It is concluded that signaling of grp78 gene transcription can occur independently of eIF-2 alpha phosphorylation or translational repression and that greater degrees of ER stress are required for eIF-2 alpha phosphorylation than for grp78 mRNA induction.

Brefeldin A↗
PubMed1997

Intracellular Ca2+ pool content and signaling and expression of a calcium pump are linked to virulence in Leishmania mexicana amazonesis amastigotes.

Virulent and avirulent clones of Leishmania mexicana amazonensis promastigotes or amastigotes were loaded with the fluorescent reagent fura 2/AM to measure intracellular free calcium ([Ca2+]i). When the cells were treated with the calcium ionophore ionomycin in the nominal absence of extracellular Ca2+, there was an increase of [Ca2+]i that was further elevated by addition of either NH4Cl, nigericin, or the vacuolar H+-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in L. mexicana amazonensis promastigotes and amastigotes is present in an acidic compartment rich in Ca2+ (acidocalcisome). Results indicated that more releasable Ca2+ is stored intracellularly in virulent amastigotes than in virulent promastigotes or avirulent cells of both stages. This higher amount of releasable Ca2+ was correlated with the presence of Ca2+ signals in the virulent amastigotes during invasion of macrophages. Ca2+ signals and invasion were reduced by preloading the parasites with intracellular Ca2+ chelators (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca2+-chelating analog (N-(2-methoxyphenyl)imidoacetic acid/AM). The gene encoding an organelle-type Ca2+-ATPase was cloned and sequenced and found overexpressed in virulent amastigotes as compared with all other forms. Together, these results demonstrate a significant link between expression of a Ca2+-ATPase, intracellular Ca2+ pool content and signaling, and virulence.

Amino Acid Sequence↗
PubMed1998

Relationship between intracellular calcium store depletion and calcium release-activated calcium current in a mast cell line (RBL-1).

The kinetic relationship between depletion of endoplasmic reticulum calcium stores and the activation of a calcium release-activated calcium current (Icrac) was investigated in the RBL-1 mast cell line. The inositol trisphosphate receptor activator, inositol 2,4, 5-trisphosphate ((2,4,5)IP3), the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, and the calcium ionophore, ionomycin, were used to deplete stored calcium. For (2,4,5)IP3 and thapsigargin, a significant delay was observed between the initiation of calcium store depletion and the activation of Icrac. However, for ionomycin, little or no delay was observed. This may indicate that a specialized subcompartment of the endoplasmic reticulum functions as a regulator of calcium entry and that this compartment is relatively resistant to depletion by (2,4,5)IP3 and thapsigargin but not to depletion by ionomycin. For all three calcium-depleting agents, the rate of development of Icrac, once initiated, was relatively constant, suggesting an all-or-none mechanism. However, there were also clear experimental situations in which submaximal, graded depletion of stored calcium resulted in submaximal activation of Icrac. This complex behavior could also result from the existence of a specific subcompartment of endoplasmic reticulum regulating Icrac. The kinetic behavior of this compartment may not be accurately reflected by the kinetics of calcium changes in the bulk of endoplasmic reticulum. These findings add to the growing body of evidence suggesting specialization of the endoplasmic reticulum calcium stores with regard to the control of capacitative calcium entry.

Animals↗
PubMed1999

Role of the inositol 1,4,5-trisphosphate receptor in Ca(2+) feedback inhibition of calcium release-activated calcium current (I(crac)).

We examined the activation and regulation of calcium release-activated calcium current (I(crac)) in RBL-1 cells in response to various Ca(2+) store-depleting agents. With [Ca(2+)](i) strongly buffered to 100 nM, I(crac) was activated by ionomycin, thapsigargin, inositol 1,4,5-trisphosphate (IP(3)), and two metabolically stable IP(3) receptor agonists, adenophostin A and L-alpha-glycerophospho-D-myoinositol-4,5-bisphosphate (GPIP(2)). With minimal [Ca(2+)](i) buffering, with [Ca(2+)](i) free to fluctuate I(crac) was activated by ionomycin, thapsigargin, and by the potent IP(3) receptor agonist, adenophostin A, but not by GPIP(2) or IP(3) itself. Likewise, when [Ca(2+)](i) was strongly buffered to 500 nM, ionomycin, thapsigargin, and adenophostin A did and GPIP(2) and IP(3) did not activate detectable I(crac). However, with minimal [Ca(2+)](i) buffering, or with [Ca(2+)](i) buffered to 500 nM, GPIP(2) was able to fully activate detectable I(crac) if uptake of Ca(2+) intracellular stores was first inhibited. Our findings suggest that when IP(3) activates the IP(3) receptor, the resulting influx of Ca(2+) quickly inactivates the receptor, and Ca(2+) is re-accumulated at sites that regulate I(crac). Adenophostin A, by virtue of its high receptor affinity, is resistant to this inactivation. Comparison of thapsigargin-releasable Ca(2+) pools following activation by different IP(3) receptor agonists indicates that the critical regulatory pool of Ca(2+) may be very small in comparison to the total IP(3)-sensitive component of the endoplasmic reticulum. These findings reveal new and important roles for IP(3) receptors located on discrete IP(3)-sensitive Ca(2+) pools in calcium feedback regulation of I(crac) and capacitative calcium entry.

Adenosine↗
PubMed2002

Inhibition of interleukin-1beta -induced NF-kappa B activation by calcium/calmodulin-dependent protein kinase kinase occurs through Akt activation associated with interleukin-1 receptor-associated kinase phosphorylation and uncoupling of MyD88.

Calcium/calmodulin-dependent protein kinase kinase (CaMKK) and Akt are two multifunctional kinases involved in many cellular responses. Although Akt and Ca(2+) signals have been implicated in NF-kappaB activation in response to certain stimuli, these results are still controversial, and the mechanism(s) involved remains unknown. In this study, we show the roles that CaMKK and Akt play in regulating interleukin-1beta (IL-1beta)-induced NF-kappaB signaling. In human embryonic kidney 293 cells, IL-1beta induces IkappaB kinase beta (IKKbeta) activation, IkappaBalpha degradation, NF-kappaB transactivation, and weak Akt activation. A CaMKK inhibitor (KN-93) and phosphatidylinositol 3-kinase inhibitors (wortmannin and LY294002) do not inhibit IL-1beta-induced NF-kappaB activation. However, IL-1beta-induced NF-kappaB activity is attenuated by increased intracellular calcium in response to ionomycin, UTP, or thapsigargin or by overexpression of CaMKKc and/or Akt. Ionomycin and CaMKKc overexpression increases Akt phosphorylation on Thr(308) and enzyme activity. Under these conditions or upon overexpression of wild type Akt, IL-1beta-induced IKKbeta activity is diminished. Furthermore, a dominant negative mutant of Akt abolishes IKKbeta inhibition by CaMKKc and ionomycin, suggesting that Akt acts as a mediator of CaMKK signaling to inhibit IL-1beta-induced IKK activity at an upstream target site. We have also identified a novel interaction between CaMKK-stimulated Akt and interleukin-1 receptor-associated kinase 1 (IRAK1), which plays a key role in IL-1beta-induced NF-kappaB activation. CaMKKc and Akt overexpression decreases IRAK1-mediated NF-kappaB activity and its association with MyD88 in response to IL-1beta stimulation. Furthermore, CaMKKc and Akt overexpression increases IRAK1 phosphorylation at Thr(100), and point mutation of this site abrogates the inhibitory effect of Akt on IRAK1-mediated NF-kappaB activation. Taken together, these results indicate a novel regulatory mechanism for IL-1beta signaling and suggest that CaMKK-dependent Akt activation inhibits IL-1beta-induced NF-kappaB activation through interference with the coupling of IRAK1 to MyD88.

Adaptor Proteins, Signal Transducing↗
PubMed2002

The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells.

The E-cadherin protein mediates Ca(2+)-dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad(100)) in prostate and mammary epithelial cells. E-cad(100) was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad(100) induction by ionomycin. Immunoblotting for spectrin and mu-calpain confirmed calpain activation in response to ionomycin treatment. Both the mu- and m-isoforms of calpain efficiently generated E-cad(100) in vitro. The E-cad(100) fragment was unable to bind to beta-catenin, gamma-catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the beta- and gamma-catenin binding motifs of E-cadherin. Because E-cadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad(100) accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which E-cadherin is functionally inactivated through calpain-mediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.

Amino Acid Sequence↗
PubMed2005

The Ca2+/calmodulin-dependent protein kinase kinases are AMP-activated protein kinase kinases.

The AMP-activated protein kinase (AMPK) is an important regulator of cellular metabolism in response to metabolic stress and to other regulatory signals. AMPK activity is absolutely dependent upon phosphorylation of AMPKalphaThr-172 in its activation loop by one or more AMPK kinases (AMPKKs). The tumor suppressor kinase, LKB1, is a major AMPKK present in a variety of tissues and cells, but several lines of evidence point to the existence of other AMPKKs. We have employed three cell lines deficient in LKB1 to study AMPK regulation and phosphorylation, HeLa, A549, and murine embryo fibroblasts derived from LKB(-/-) mice. In HeLa and A549 cells, mannitol, 2-deoxyglucose, and ionomycin, but not 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), treatment activates AMPK by alphaThr-172 phosphorylation. These responses, as well as the downstream effects of AMPK on the phosphorylation of acetyl-CoA carboxylase, are largely inhibited by the Ca(2+)/ calmodulin-dependent protein kinase kinase (CaMKK) inhibitor, STO-609. AMPKK activity in HeLa cell lysates measured in vitro is totally inhibited by STO-609 with an IC50 comparable with that of the known CaMKK isoforms, CaMKKalpha and CaMKKbeta. Furthermore, 2-deoxyglucose- and ionomycin-stimulated AMPK activity, alphaThr-172 phosphorylation, and acetyl-CoA carboxylase phosphorylation are substantially reduced in HeLa cells transfected with small interfering RNAs specific for CaMKKalpha and CaMKKbeta. Lastly, the activation of AMPK in response to ionomycin and 2-deoxyglucose is not impaired in LKB1(-/-) murine embryo fibroblasts. These data indicate that the CaMKKs function in intact cells as AMPKKs, predicting wider roles for these kinases in regulating AMPK activity in vivo.

AMP-Activated Protein Kinase Kinases↗
PubMed2002

SYK and LYN mediate B-cell receptor-independent calcium-induced apoptosis in DT-40 lymphoma B-cells.

Here, we report that the calcium ionophore ionomycin induces a massive Ca2+-dependent apoptosis in wildtype DT-40 chicken B lymphoma cells, as well as in BTK-deficient, PLCgamma2-deficient and IP3 receptor-deficient DT-40 cells, but not in LYN- or SYK-deficient DT-40 cells. Notably, the deficiency of CSK, a negative regulator of Src-family PTK, promoted ionomycin-induced apoptosis of DT-40 cells. Reconstitution of SYK-deficient cells with wild-type SYK restored the apoptotic response of the cells to ionomycin, but the expression of FYN or LCK in LYN-deficient cells did not restore the apoptotic response of LYN-deficient cells. Taken together, our data suggests that both LYN and SYK, but not BTK, FYN or LCK, are crucial mediators of BCR-independent Ca2+-induced apoptosis in DT-40 lymphoma B cells.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1991

Granulocyte/macrophage colony-stimulating factor and interleukin 3 release from human peripheral blood eosinophils and neutrophils.

Human peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin. The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A. The survival-enhancing activity was completely abolished by treatment with anti-interleukin 3 (IL-3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies. Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL-3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced IL-3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms.

Cell Survival↗
PubMed1990

Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role.

To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free [Ca] ([Ca]i) and the potassium conductance (gK) of the basolateral membrane in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value ([Ca]'i) normalized to control. To assess changes in basolateral gK, we measured the short circuit current (Isc) in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K+ gradient (Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986. J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in [Ca]'i and gK. The carbachol-induced changes in gK appeared to be dependent on the increase in [Ca]i because stimulation of gK was significantly diminished when the hormone-induced increase in [Ca]'i was blunted, either by loading the cells with BAPTA or by reducing the extracellular [Ca]. The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state [Ca]'i. The changes in gK and [Ca]'i after stimulation with carbachol were correlated and ionomycin also increased gK and [Ca]'i in a parallel manner. The carbachol-induced delta gK per delta[Ca]'i, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gK.

Amphotericin B↗
PubMed2001

Glucose and pyruvate metabolism of preimplantation goat blastocysts following in vitro fertilization and parthenogenetic activation.

The energy metabolism of preimplantation embryos can be used to predict viability and postimplantation development. Although preimplantation development and mean blastocyst cell numbers of goat in vitro-fertilized (IVF) embryos and chemically activated parthenogenotes are comparable, mammalian parthenogenotes are not viable, with most dying shortly after implantation. The objective of this study was to compare glucose and pyruvate metabolism of IVF goat blastocysts with that of parthenogenetic blastocysts developing from chemically activated oocytes. Embryos derived from IVF and parthenogenotes produced by exposing oocytes to either ionomycin or ethanol followed by 6-dimethylaminopurine (6-DMAP) were cultured in G1.2/G2.2 sequential culture media. Metabolism was determined for individual blastocysts using [5-3H]glucose and [2-14C]pyruvate to determine glycolytic and Kreb's cycle activity, respectively. Data were analyzed by ANOVA. A significantly higher percentage of activated oocytes underwent cleavage and developed to the blastocyst stage compared to IVF oocytes (p < 0.05). There was no significant difference in glucose or pyruvate metabolism between IVF and parthenogenetically activated blastocysts. Mean glucose metabolism through glycolysis was 154.9 +/- 29.1, 130.3 +/- 17.1, and 129 +/- 16.5 pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Mean pyruvate metabolism through the Kreb's cycle was 28.1 +/- 8.0, 15.8 +/- 4.2, and 24.4 +/- 4.4 in pmol/embryo/3 h for IVF, ethanol-activated, and ionomycin-activated blastocysts, respectively. Our results suggest that known differences in postimplantation development observed in IVF versus parthenogenetic embryos cannot be attributed to differences in pyruvate or glucose metabolism in the preimplantation blastocysts. Thus, these activation protocols result in embryos capable of appropriate regulation of key metabolic enzymes.

Adenine↗
PubMed1996

In vitro differentiation and commitment of CD4+ CD8+ thymocytes to the CD4 lineage, without TCR engagement.

Thymocyte positive selection is based on protection of immature CD4/CD8 double-positive (DP) thymocytes from apoptosis and their differentiation into CD4 or CD8 single-positive (SP) cells. Intracellular signals essential for positive selection appear to be induced through the TCR and some of the accessory molecules including LFA-1, CD4 and CD8 upon interaction with thymic stromal cells. The signals, however, still remain to be identified. Since physiological levels of glucocorticoids potentially induce or enhance thymocyte apoptosis even in vivo, the signals are likely to inhibit the apoptotic effect of glucocorticoids. We have previously shown that proper cross-linking of TCR-CD3 with LFA-1, CD4 or CD8 inhibited glucocorticoid-induced thymocyte apoptosis in vitro, and that a proper combination of the calcium ionophore, ionomycin and the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), mimicked the inhibitory effect. Here we determined whether this combination of ionomycin and PMA induces differentiation of isolated DP thymocytes from normal and TCR transgenic mice. We found that pretreatment of DP thymocytes with ionomycin and PMA followed by 1 day culture of the cells without the reagents resulted in the differentiation of the cells into CD4 SP and CD4+ CD8lo T cells that have mostly committed to the CD4 lineage. The changes in expression of other differentiation markers were also in good accordance with those associated with positive selection, except the final maturation. The results indicate that moderate and transient increases in intracellular Ca2+ level and PKC activity induce differentiation and commitment of DP thymocytes to the CD4 lineage, and suggested that the biochemical pathway leading to positive selection is based on a similar mechanism.

Animals↗
PubMed2003

Decreased CD154 expression by neonatal CD4+ T cells is due to limitations in both proximal and distal events of T cell activation.

Neonatal CD4(+) T cells express less CD154 protein and mRNA than adult CD4(+) T cells after activation by calcium ionophore and phorbol ester, but the mechanism for this reduced expression and its relevance to the primary immune response remain unclear. We compared expression of CD154 protein and mRNA and CD154 gene promoter activity by purified naive (CD45RA(high)CD45RO(low)) neonatal and adult CD4(+) T cells after activation by calcium ionophore (ionomycin) and phorbol myristate acetate (PMA) treatment or by engagement of alphabeta TCR-CD3 complex. Substantial and consistent reductions in expression by neonatal cells were found in all cases and were paralleled by decreased CD154-dependent activation of a B cell line. CD69 expression by neonatal CD4(+) T cells after alphabeta TCR-CD3 engagement was also reduced compared to adult cells, which suggested that limitations in activation-induced signaling by neonatal CD4(+) T cells occurred at a point upstream of where the signaling pathways leading to CD154 and CD69 expression diverge. Decreased CD154 expression by neonatal cells after alphabeta TCR-CD3 engagement was paralleled by a lower free intracellular calcium concentration, a key event for CD154 gene transcription. Reduced CD154 promoter activity by neonatal cells persisted when proximal signaling events were bypassed using ionomycin and PMA, suggesting an additional and more distal mechanism for decreased transcription. In contrast, CD154 mRNA stability was similar in neonatal and adult cells after either ionomycin and PMA stimulation or engagement of the alphabeta TCR-CD3 complex. We conclude that decreased CD154 production by neonatal CD4(+) T cells is due to limitations in both proximal and distal activation events, which together ultimately limit CD154 gene transcription.

Adult↗
PubMed1991

The effects of elevation and depletion of intracellular free calcium on progesterone and prostaglandin production by the primate corpus luteum.

The role of the phosphatidylinositol second messenger system in luteal regulation has not been extensively studied, particularly in the primate. The objectives of this study were (1) to further characterize the response of the primate CL to the calcium ionophore A23187, in terms of intracellular free calcium concentrations ([Ca2+]i) and progesterone (P) production; and (2) to assess the effects of depleting, as well as elevating, available calcium on luteal P and prostaglandin (PG) production. The response to A23187, in terms of [Ca2+]i, was measured by fura-2 fluorescence microscopy of single small and large luteal cells. A23187 significantly increased [Ca2+]i in both cell types (p less than 0.01). P production (basal and hCG-stimulated) by dispersed primate luteal cells incubated for various times (1-8 h) with and without A23187 was measured. Treatment with A23187 rapidly (within 1-2 h) attenuated (p less than 0.05) the time-dependent increase in basal and hCG-stimulated P production. Luteal P and PG production following treatment with the calcium ionophore, ionomycin, alone or in combination with additional CaCl2, was also monitored. Treatment with ionomycin (p less than 0.01) and CaCl2 (p less than 0.01) inhibited luteal P production. In contrast, treatment with ionomycin stimulated (p less than 0.01) luteal PG production. To determine the effects of Ca2+ depletion on luteal function, P and PG production by cells incubated for 2 and 8 h in the absence and presence of the Ca(2+)-chelator EGTA was measured. Luteal production of both P and PG was inhibited by 8-h treatment with EGTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Regional differences in bioelectrical properties and anion secretion in cultured epithelia from rat and human male excurrent ducts.

Bioelectrical properties and anion secretion in cultured epithelia from different regions of rat and human male excurrent ducts were studied by measuring the short-circuit currents (ISC). In all regions of the rat excurrent duct, Cl- secretion accounts for over 90% of the basal ISC, although the magnitude varied in different regions. Cl- secretion was found to be mediated by a Cl-/HCO3- exchanger, an Na+/H+ exchanger, and an Na+/K+/2Cl- symport located on the basolateral side of the epithelial cells. Forskolin, an activator of adenylate cyclase, and ionomycin, a Ca2+ ionophore, were used to investigate the relative importance of cAMP and Ca2+ as intracellular messengers regulating Cl- secretion in different regions. It was found that in both species, the forskolin-evoked ISC response was larger in the proximal end (efferent duct/caput epididymidis [rat/human, respectively]) than in the distal end (cauda/corpus epididymidis). The response to ionomycin in the rat cauda epididymidis (distal end) was larger than that in the efferent duct (proximal end); on the other hand, no significant difference in the ionomycin-induced ISC was observed in the caput and the corpus regions from the human epididymis. Our results indicate that while the cAMP- and Ca(2+)-dependent pathways are both involved in regulating Cl- secretion in all regions along the male excurrent ducts in both species, a regional difference exists with respect to the relative importance of the two regulatory pathways involved in Cl- secretion along the male reproductive tract.

Animals↗
PubMed2001

Parthenogenetic activation of rhesus monkey oocytes and reconstructed embryos.

This study determines the efficiency of sequential calcium treatments (electroporation or ionomycin) combined with protein synthesis (cycloheximide) or phosphorylation inhibitors (6-dimethylaminopurine) or the specific maturation promoting factor (MPF) inhibitor, roscovitine, in inducing artificial activation and development of rhesus macaque parthenotes or nuclear transfer embryos. Exposure of oocytes arrested at metaphase II (MII) to ionomycin followed by 6-dimethylaminopurine or to electroporation followed by cycloheximide and cytochalasin B induced pronuclear formation and development to the blastocyst stage at a rate similar to control embryos produced by intracytoplasmic sperm injection. Parthenotes did not complete meiosis or extrude a second polar body, consistent with their presumed diploid status. In contrast, oocytes treated sequentially with ionomycin and roscovitine extruded the second polar body and formed a pronucleus at a rate higher than that observed in controls. Following reconstruction by nuclear transfer, activation with ionomycin/6-dimethylaminopurine resulted in embryos that contained a single pronucleus and no polar bodies. All nuclear transfer embryos activated with ionomycin/roscovitine contained one large pronucleus. However, a third of these embryos emitted one or two polar bodies, clearly containing chromatin material. In summary, we have identified simple yet effective methods of oocyte or cytoplast activation in the monkey, ionomycin/6-dimethylaminopurine, electroporation/cycloheximide/cytochalasin B, and ionomycin/roscovitine, which are applicable to parthenote or nuclear transfer embryo production.

Animals↗
PubMed1992

Bryostatin 1 activates T cells that have antitumor activity.

Several strategies have been used to stimulate the growth of tumor-specific T cells in place of tumor antigen. One approach is to use pharmacologic agents to activate the second messenger pathways of T-cell activation. In the present study, we examined the ability of the protein kinase C activator bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to stimulate the growth of lymphocytes obtained from the axillary lymph nodes (DLN) draining a progressively growing intradermal plasmacytoma tumor. Draining lymph node cells were initially cultured with autologous tumor cells and 20 U/ml of interleukin-2 (IL-2) for 7 days. The lymphocytes were then incubated with various concentrations of bryostatin 1 plus 1 microM ionomycin and cultured for an additional 14 days in IL-2. DLN cells initially cultured with autologous tumor and then restimulated with 5 nM bryostatin 1 and 1 microM ionomycin exhibited marked in vitro proliferation and 15-fold expansion of cell numbers over 2 weeks. The cells expanded with B/I were predominantly CD8+ T cells and retained specific in vitro cytotoxicity against autologous tumor. When adoptively transferred to mice with established liver metastases, DLN cells restimulated with B/I-mediated specific tumor regression.

Animals↗
PubMed1999

Inhibition of intracellular Ca2+ mobilisation by low antiproliferative concentrations of thapsigargin in human vascular smooth-muscle cells.

Low nanomolar concentrations of thapsigargin, a modulator of intracellular Ca2+ ([Ca2+]i) pools, inhibit vascular smooth-muscle cell (VSMC) proliferation. Because the mechanisms underlying this effect have not been defined, the effect of antiproliferative concentrations of thapsigargin on [Ca2+]i in fura-2-loaded VSMCs was studied by using dynamic video imaging of [Ca2+]i. After seeding on coverslips, human VSMCs were incubated for 1-48 h with thapsigargin before loading with fura-2 or during imaging. Mobilisation of [Ca2+]i was stimulated with 1 microM ionomycin in Ca2+-free medium and the increase in [Ca2+]i detected by using Ca2+ imaging techniques. Continuous exposure of cells to low concentrations of thapsigargin (which failed measurably to increase in [Ca2+]i) reduced the ionomycin response in a time- and dose-dependent manner (100% inhibition at 10 nM thapsigargin after 1 h exposure). After exposure of cells to 10 nM thapsigargin for 1 h followed by washing and further incubation for < or = 72 h, there was a time-dependent recovery of the ionomycin response. Because the concentrations of thapsigargin and exposure times are identical to those that inhibit replication in VSMCs, it is proposed that depletion of [Ca2+]i pools mediates the inhibitory effect of thapsigargin on VSMC proliferation.

Calcium↗