• Eicosanoid-mediated regulation of intracellular calcium pools and cell growth

      Graber, Matthew Noah; Gill, Donald L. (1996)
      The inositol 1,4,5-trisphosphate (InsP{dollar}\sb3{dollar})-sensitive Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools of {dollar}\rm DDT\sb1MF{lcub}-{rcub}2{dollar} smooth muscle cells empty after inhibition of the intracellular Ca{dollar}\sp{lcub}2+{rcub}{dollar}-ATPase by thapsigargin. Pool emptying causes cells to cease division and enter a stable, quiescent G{dollar}\sb0{dollar}-like state. High serum treatment of these pool-depleted quiescent cells induces the reappearance of functional Ca{dollar}\sp{lcub}2+{rcub}{dollar} pump protein, re-filling of InsP{dollar}\sb3{dollar}-sensitive Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools, and re-entry of cells into the cell cycle. This recovery is mediated by the direct donation of essential fatty acids from the high serum to the pool-depleted cells. The essential fatty acids linolenic acid, linoleic acid, and arachidonic acid each induced recovery of Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools and re-entry of cells into the cell cycle with an EC{dollar}\sb{lcub}50{rcub}{dollar} of approximately 5 {dollar}\mu{dollar}M; the action of each was dependent on protein synthesis. All non-essential fatty acids and growth factors tested did not promote recovery. Inhibitors of the prostanoid and lipoxygenase metabolism pathways had no effect on essential fatty acid-induced Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool or growth recovery. However, the cytochrome P-450 inhibitors SKF-525A, metyrapone, and nordihydroguaiaretic acid each prevented the action of AA. Importantly, treatment of quiescent cells with either of 8,9- or 11,12-epoxyeicosatrienoic acid (EET), two of the four regiospecific cytochrome P-450 metabolites of arachidonic acid, also induced recovery. However, further metabolism of the effective EETs either to dihydroxyeicosatrienoic acid metabolites or through subsequent eicosanoid synthesizing pathways appears to be unnecessary. Evidence suggests that neither protein kinase activity nor the participation of cGMP or cAMP is involved in the EET-induced recovery process. These results are important in understanding the role of cytochrome P-450 derived eicosanoids in cellular regulation and the relationship between pool emptying and cell cycle control.
    • Identification of intracellular calcium pools and calcium translocation mechanisms in DDT(1)MF-2 smooth muscle cells

      Bian, Junhui; Gill, Donald L. (1992)
      The focus of the present studies is on the functional characterization of discrete Ca{dollar}\sp{lcub}2+{rcub}{dollar} pools on the basis of subcellular location, Ca{dollar}\sp{lcub}2+{rcub}{dollar} transport properties, and functional roles. In both saponin-permeabilized DDT{dollar}\sb1{dollar}MF-2 smooth muscle cells and purified rough endoplasmic reticulum (ER) vesicles from these cells, sphingosine derivatives, in particular, sphingosine (Sph) and sphingosyl-phosphorylcholine (SPC) were observed to induce rapid and profound intracellular Ca{dollar}\sp{lcub}2+{rcub}{dollar} release as revealed by {dollar}\sp{lcub}45{rcub}{dollar}Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake and release assays. The properties of sphingoid base-induced Ca{dollar}\sp{lcub}2+{rcub}{dollar} release were found highly comparable to their reported effects on PKC inhibition in terms of sensitivity, molecular specificity and reversibility. Sphingoid bases released Ca{dollar}\sp{lcub}2+{rcub}{dollar} up to the same level of the combined action of inositol-1,4,5-triphosphate (InsP{dollar}\sb3){dollar} and GTP, and no further Ca{dollar}\sp{lcub}2+{rcub}{dollar} release induced by InsP{dollar}\sb3{dollar} together with GTP was seen after maximal effect of sphingosine derivatives, indicating a shared Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool target with InsP{dollar}\sb3{dollar} and GTP. The action of Sph, in contrast to that of SPC, was blocked by ADP (100 {dollar}\mu{dollar}M) addition, ATP depletion and low temperature, indicating that Sph had no intrinsic activity in releasing Ca{dollar}\sp{lcub}2+{rcub}{dollar} and must be converted into an active form. The results indicate that generation of sphingoid bases in cells may activate a dual signaling pathway involving Ca{dollar}\sp{lcub}2+{rcub}{dollar} release in parallel to PKC inhibition. Thapsigargin (TG) inhibited 75% of total intracellular ATP-dependent Ca{dollar}\sp{lcub}2+{rcub}{dollar} uptake with an IC{dollar}\sb{lcub}50{rcub}{dollar} of 30 nM. TG-sensitive Ca{dollar}\sp{lcub}2+{rcub}{dollar} pumps exist in two previously defined pools--the InsP{dollar}\sb3{dollar}-sensitive, oxalate-permeable Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool, and the InsP{dollar}\sb3{dollar}-insensitive, oxalate-impermeable Ca{dollar}\sp{lcub}2+{rcub}{dollar} pool. A brief TG treatment of 30 min caused persistent Ca{dollar}\sp{lcub}2+{rcub}{dollar} depletion in TG-sensitive pools as well as total arrest of cell growth. A precise correlation between the Ca{dollar}\sp{lcub}2+{rcub}{dollar} content in signaling pools and cell growth was shown to exist. (Abstract shortened by UMI.)
    • Intracellular calcium pump expression, calcium pool function and cell growth

      Waldron, Richard Taliesin; Gill, Donald L. (1996)
      Expression of functional intracellular Ca2+{ pumps is essential for operation of Ca2+ signaling pools. The aim of these studies was to detect functional pumps within Ca2+ pumping pools and identify the Ca2+ transport properties and specific Ca2+ signaling functions of these pools. (1) Using DDT{dollar}\sb1{dollar}MF-2 smooth muscle cells growth-arrested by exposure to the intracellular Ca2+ pump blocker thapsigargin (TG) at 3 {dollar}\mu{dollar}M for 24 hr, treatment with 20% serum for 6 hr without TG induced expression of functional Ca2+ pump protein detected as a 110 kDa TG-sensitive phosphorylated intermediate; 2.5% serum treatment resulted in no functional pump expression. Western analysis revealed only a slight serum-induced increase in total Ca2+ pump protein. The timing of appearance of new functional Ca2+ pump protein (first detected within 1 hr of high serum treatment) and the required duration of the high serum treatment (approx. 35 min.) for subsequent full recovery of Ca2+ pools and growth indicate the necessity of functional endoplasmic reticulum Ca2+ pumps in serum-induced cell growth and reflect a precise signaling period during which quiescent cells commit to a progression of events including Ca2+ pump expression, Ca2+ pool function, reentry into the cell cycle, and cell division. (2) Further studies utilized TG-resistant DC-3F/TG2 cells cultured with 2 {dollar}\mu{dollar}M TG; these cells have a doubling time (24 hr) not significantly different from parent DC-3F Chinese hamster lung cells without TG. TG inhibited {dollar}\rm \sp{lcub}45{rcub}Ca\sp{lcub}2+{rcub}{dollar} uptake of two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG within permeabilized parent DC-3F cells; the IC{dollar}\sb{lcub}50{rcub}{dollar} values for TG were 200 pM and 4 {dollar}\mu{dollar}M, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells were similar (0.23 {dollar}\pm{dollar} 0.10 and 0.18 {dollar}\pm{dollar} 0.08 nmol Ca{dollar}\sp{lcub}2+{rcub}{dollar}/10{dollar}\sp6{dollar} cells, respectively), yet in DC-3F/TG2 cells, only TG-insensitive pumps were functional. In both cell types, each Ca2+ pump activity (regardless of TG-sensitivity) had high Ca2+ affinity (K{dollar}\sb{lcub}\rm m{rcub}{dollar} values {dollar}\simeq{dollar} 0.1 {dollar}\mu{dollar}M), and similar ATP-dependence and vanadate-sensitivity, In DC-3F cells, sensitivity to release with InsP{dollar}\sb3{dollar} or GTP and oxalate-permeability were exclusive to the TG-sensitive Ca2+ pool. GTP-induced Ca2+ uptake in the presence of oxalate indicated Ca2+ transfer between distinct pools in the DC-3F cells. In resistant DC-3F/TG2 cells, almost 50% of total TG-insensitive Ca2+ accumulation was releasable with InsP{dollar}\sb3{dollar}; unlike the parent cells this pool was not oxalate-permeable. These findings reveal that the TG-insensitive Ca2+ pumping pool within resistant DC-3F/TG2 cells contains functional InsP{dollar}\sb3{dollar} receptors. Measurements of cytosolic Ca2+ concentrations ((Ca2+{rcub}\rbrack\sb{lcub}\rm i{rcub}){dollar} within intact, fura-2 loaded parent DC-3F cells showed typical influx of Ca{dollar}\sp{lcub}2+{rcub}{dollar} through store-operated channels (SOCs) after TG-induced pool depletion. Unlike the parent DC-3F cells, DC-3F/TG2 cells had constitutively activated basal Ca2+ entry through SOCs which could be rapidly increased by transient external Ca2+ removal; thus, the machinery for activating Ca2+ entry is continuously operational. The effect of oleoyl lysophosphatidic acid (LPA) to release Ca2+ pools within DC-3F cells by interaction with receptors linked to generation of InsP{dollar}\sb3{dollar} was abolished by prior TG-addition. (Abstract shortened by UMI.)
    • Modification of calcium pool function by fatty acids and their coenzyme A esters

      Rys-Sikora, Krystyna Ewa; Gill, Donald L. (1997)
      Intracellular Ca2+ pools are essential elements in the generation of Ca2+ signals within cells, however, their nature and identity have remained elusive. Ca2+ pools are complex and dynamic entities and are modified by G protein-induced membrane fusion events, allowing GTP-activated transfer of Ca2+ between discrete Ca2+ pools. Studies examined the modification of GTP-activated Ca2+ translocation process by fatty acyl-CoA esters and fatty acids since G protein action can be modified by fatty acylation. Using permeabilized DDT1MF-2 smooth muscle cells, palmitoyl-CoA (IC50 = 0.5 muM) was observed to completely block 45Ca2+ release activated by GTP, while having no effect on InsP3-induced Ca2+ release. Fatty acyl chain length was important, only C-13 to C-16 fatty acyl-CoA esters fully inhibited the action of GTP. CoA(10 muM) also blocked GTP-activated Ca2+ release, although the free sulfhydryl group and ATP requirements indicated that CoA must be fatty acylated to be effective. The nonhydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, blocked the GTP effect identically to myristoyl- and palmitoyl-CoA. Thus, fatty acyl transfer is not required indicating that the blockade is due to a direct allosteric modification of a component of the GTP-activated process. Palmitoyl-CoA not only inhibited but completely reversed GTP-activated Ca2+ release. In the presence of oxalate, GTP-activated Ca2+ transfer causes a substantial increase in Ca2+ accumulation; palmitoyl-CoA also completely reversed this effect. These results provide strong evidence that GTP-activated Ca2+ translocation does not reflect a full fusion event, but the formation of a reversible prefusion pore. The actions of fatty acids were very different from their acyl-CoA esters; 10-100 muM palmitate (C16:0) had a major stimulatory effect on GTP-mediated Ca2+accumulation. The biphasic nature of the palmitate effect was characteristically similar to the effect of oxalate; however, the EC50 for palmitate was 20 muM (approx. 100-fold lower that of oxalate). This activation was highly specific for chain length and degree of saturation. Only pentadecanoic acid (C15:0) duplicated this effect, unsaturated fatty acids were completely ineffective. Both palmitate- and oxalate-activated Ca2+ accumulation in the presence of GTP were inhibited by the anion transport inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS). Hence, both Ca2+-complexing agents may enter anion-permeable Ca2+ subpools through similar anion channels. To further examine the nature of these Ca2+ complexes, a comparison of the releasability of Ca2+ using InsP3 and the Ca2+ ionophore, A23187 was undertaken. In the presence of oxalate, GTP-mediated accumulation of Ca2+ was only slowly releasable by InsP3 or A23187. Whereas, Ca2+ accumulated in the presence of palmitate and GTP was completely releasable by A23187, only a small fraction of the accumulated Ca2+was released by InsP3. These data suggest important differences between the state and possibly, the location of oxalate- and palmitate-Ca2+ complexes within Ca2+ pools. Thus, the formation of Ca2+-fatty acid complexes and, in turn, the activation of Ca2+ accumulation may reflect a major physiological role for fatty acids in stabilizing Ca2+ within the lumen of Ca2+ pools.
    • Regulation of calcium entry mechanisms by intracellular calcium pools

      Ufret-Vincenty, Carmen Angeles; Gill, Donald L. (1998)
      Release of Ca2+ from intracellular pools is the main trigger for activation of Ca2+ entry during the generation of Ca2+ signals in non-excitable cells. The aim of these studies is to investigate the relationship between Ca2+ pool emptying and activation of Ca2+ influx. Using the smooth muscle cell line, DDT1MF-2, changes in cytosolic Ca2+ concentration were monitored by loading cells with the fluorescent Ca2+ indicator, fura-2. The studies reveal that Ca2+ pool depletion by treatment with the SERCA pump inhibitors, thapsigargin and 2,5-Di-tert-butylhydroquinone (DBHQ), stimulates the activation of a novel and distinct Ca2+ entry pathway sensitive to caffeine. Refilling of Ca2+ pools occurs concomitantly with the disappearance of the caffeine-sensitive Ca2+ entry pathway. Stimulation of the caffeine-sensitive Ca2+ entry pathway occurs independently of the means used to deplete intracellular Ca2+ pools (pump inhibition or ionophore-induced depletion). Therefore, activation of the caffeine-sensitive Ca2+ entry pathway is directly controlled by Ca2+ pool depletion. Ion permeability studies indicate that the Ca2+ entry pathway sensitive to caffeine is less selective for Ca2+ than store-operated Ca2+ entry, the major Ca2+ influx pathway activated by pool depletion in a wide variety of cells. Nitric oxide-induced thiol modification (nitrosylation) has recently been shown to have major modulatory effects on the activity of several Ca2+ channels. Therefore, it was important to investigate the role of thiol nitrosylation in controlling Ca2+ entry and its activation by pool depletion. Studies reveal that nitric oxide donors activate a substantial entry of Ca2+ through a direct pathway which is independent of guanylate cyclase activation, a well-studied target for nitric oxide. Cell permeant alkylating agents activate an entry of Ca2+ remarkably similar to nitric oxide-induced Ca2+ entry, indicating that activation of Ca2+ entry relies on modification of one or more thiol residues in the channel or a closely associated protein. Most significantly, Ca2+ pool emptying strongly stimulates the Ca2+ entry activated by both nitric oxide donors and alkylating agents, revealing a direct link between thiol nitrosylation and activation of a store-sensitive Ca2+ entry pathway. These studies provide further evidence for the strong involvement of Ca2+ pool content in controlling the activity of major Ca2+ entry mechanisms.