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D L Gill

Publications and source records attributed to D L Gill.

At least 37 records · Page 2Linked to original sources
PubMed1996

Calcium pools, calcium entry, and cell growth.

The Ca2+ pump and Ca2+ release functions of intracellular Ca2+ pools have been well characterized. However, the nature and identity of Ca2+ pools as well as the physiological implications of Ca2+ levels within them, have remained elusive. Ca2+ pools appear to be contained within the endoplasmic reticulum (ER); however, ER is a heterogeneous and widely distributed organelle, with numerous other functions than Ca2+ regulation. Studies described here center on trying to determine more about subcellular distribution of Ca2+ pools, the levels of Ca2+ within Ca2+ pools, and how these intraluminal Ca2+ levels may be physiologically related to ER function. Experiments utilizing in situ high resolution subcellular morphological analysis of ER loaded with ratiometric fluorescent Ca2+ dyes, indicate a wide distribution of inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools within cells, and large changes in the levels of Ca2+ within pools following Insp3-mediated Ca2+ release. Such changes in Ca2+ may be of great significance to the translation, translocation, and folding of proteins in ER, in particular with respect to the function of the now numerously described luminal Ca(2+)-sensitive chaperonin proteins. Studies have also focussed on the physiological role of pool Ca2+ changes with respect to cell growth. Emptying of pools using Ca2+ pump blockers can result in cells entering a stable quiescent G(o)-like growth state. After treatment with the irreversible pump blocker, thapsigargin, cells remain in this state until they are stimulated with essential fatty acids whereupon new pump protein is synthesized, functional Ca2+ pools return, and cells re-enter the cell cycle. During the Ca2+ pool-depleted growth-arrested state, cells express a Ca2+ influx channel that is distinct from the store-operated Ca2+ influx channels activated after short-term depletion of Ca2+ pools. Overall, these studies indicate that significant changes in intraluminal ER Ca2+ do occur and that such changes appear linked to alteration of essential ER functions as well as to the cell cycle-state and the growth of cells.

Animals↗
PubMed1995

A novel Ca2+ entry mechanism is turned on during growth arrest induced by Ca2+ pool depletion.

Ca2+ pool depletion with Ca2+ pump blockers induces growth arrest of rapidly dividing DDT1MF-2 smooth muscle cells and causes cells to enter a stable, quiescent G0-like growth state (Short, A.D., Bian, J., Ghosh, T.K., Waldron, R.T., Rybak, S.L., and Gill, D.L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Here we reveal that induction of this quiescent growth state with the Ca2+ pump blocker, thapsigargin, is correlated with the appearance of a novel caffeine-activated Ca2+ influx mechanism. Ca2+ influx through this mechanism is clearly distinct from and additive with Ca2+ entry through store-operated channels (SOCs). Whereas SOC-mediated entry is activated seconds after Ca2+ pool release, caffeine-sensitive influx requires at least 30 min of pool emptying. Although activated in the 1-10 mM caffeine range, this mechanism has clearly distinct methylxanthine specificity from ryanodine receptors and is not modified by ryanodine. It is also unaffected by the Ca2+ channel blockers SKF96365 or verapamil and is independent of modifiers of cyclic nucleotide levels. Growth arrest by thapsigargin-induced Ca2+ pool depletion can be reversed by treatment with 20% serum (Waldron, R.T., Short, A.D., Meadows, J.J., Ghosh, T.K., and Gill, D.L. (1994) J. Biol. Chem. 269, 11927-11933). The serum-induced return of functional Ca2+ pools and reentry of cells into the cell cycle correlates exactly with the disappearance of the caffeine-sensitive Ca2+ influx mechanism. Therefore, appearance and function of this novel Ca2+ entry mechanism are closely tied to Ca2+ pool function and cell growth state and may provide an important means for modifying exit from or entry into the cell cycle.

Animals↗
PubMed1995

Thapsigargin-resistant intracellular calcium pumps. Role in calcium pool function and growth of thapsigargin-resistant cells.

Exposure of cells to the intracellular Ca2+ pump blocker, thapsigargin (TG), results in emptying of Ca2+ pools and termination of cell proliferation (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). DC-3F Chinese hamster lung cells were made resistant to TG by long-term stepwise exposure to increasing TG concentrations in culture (Gutheil, J. C., Hart, S. R., Belani, C. P., Melera, P. W., and Hussain, A. (1994) J. Biol. Chem. 269, 7976-7981). Since these cells (DC-3F/TG2) grow in the presence of TG, it was important to ascertain what Ca2+ pool function they retain. TG-resistant DC-3F/TG2 cells cultured with 2 microM TG had a doubling time (24 h) not significantly different from the parent DC-3F cells without TG. Analysis of TG-induced inhibition of 45Ca2+ uptake into permeabilized parent DC-3F cells revealed two distinct Ca2+ pump activities with 20,000-fold different sensitivities to TG; the IC50 values for TG were 200 pM and 4 microM, representing 80% and 20% of total pumping activity, respectively. Total pump activity in parent DC-3F and resistant DC-3F/TG2 cells was similar (0.23 +/- 0.10 and 0.18 +/- 0.08 nmol of Ca2+/10(6) cells, respectively). In DC-3F/TG2 cells, up to 100 nM TG had no effect on Ca2+ pumping; however, almost all pumping was blocked at higher TG concentrations with an IC50 of 5 microM. In both cell types, each Ca2+ pump activity (regardless of TG sensitivity) had high Ca2+ affinity (Km values congruent to 0.1 microM) and similar ATP dependence and vanadate sensitivity. In DC-3F cells, the TG-sensitive Ca2+ pool was releasable with inositol 1,4,5-trisphosphate (InsP3) or GTP and was oxalate-permeable; the TG-insensitive pool in these cells was not InsP3-releasable. 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 InsP3; unlike the parent cells, this pool was not oxalate-permeable, and GTP induced no Ca2+ transfer between pools in the presence of oxalate. Thus, whereas InsP3 releases Ca2+ only from the high TG sensitivity Ca2+ pumping pool in parent DC-3F cells, in resistant DC-3F/TG2 cells the TG-resistant Ca2+ pumping pool now contains functional InsP3 receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Attentional strategy use by experienced distance runners: physiological and psychological effects.

The purpose of this investigation was twofold: (a) to compare most and least economical runners on use of attentional strategies and (b) to determine whether the least economical runners within the subject sample could improve running economy (RE) with the use of an active associative (relaxation) attentional strategy. Subjects (N = 36) completed an initial assessment of attentional style and RE; then the 12 least economical runners ran in each of three laboratory sessions using control, passive associative, and active associative attentional strategies. Results showed that the most economical and least economical runners did not differ in associative style use. The most economical runners, however, reported less dissociation use and more use of relaxation than did the least economical runners. No significant physiological or psychological changes were associated with any of the three attentional strategy conditions. The findings are related to possible differences in how most and least economical runners use associative strategies.

Adolescent↗
PubMed1994

Modification of GTP-activated calcium translocation by fatty acyl-CoA esters. Evidence for a GTP-induced prefusion event.

A sensitive and specific GTP-activated Ca2+ translocation process induces rapid Ca2+ movements within cells and appears to reflect G protein-induced membrane fusion or junctional communication between discrete subpopulations of Ca(2+)-pumping organelles (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Since fatty acylation can modify G protein action, modification of GTP-induced Ca2+ translocation by fatty acyl-CoA was investigated to throw light on the mechanism underlying Ca2+ transfer. Using permeabilized DDT1MF-2 smooth muscle cells, 2 microM palmitoyl-CoA completely blocked Ca2+ release activated by 20 microM GTP, while having no effect on inositol 1,4,5-trisphosphate-induced Ca2+ release. The IC50 (50% inhibitory concentration) for palmitoyl-CoA was 0.5 microM. Above 3 microM, palmitoyl-CoA inhibited Ca2+ accumulation. Fatty acyl chain length was important, C-13 to C-16 fatty acyl-CoA esters all fully blocking the action of GTP; the IC50 for myristoyl-CoA was also 0.5 microM. C-18 or larger acyl groups had diminished effectiveness as did C-8 or smaller acyl groups. Acetyl-CoA had no blocking effect. In contrast, 10 microM CoA itself blocked GTP-induced Ca2+ release. CoA required a free sulfhydryl group to block, desulfo-CoA having no effect. Removal of ATP by hexokinase and glucose prevented the action of CoA but not palmitoyl-CoA. The free sulfhydryl and ATP requirements indicated CoA was being acylated by endogenous fatty-acyl-CoA synthetase to be effective. The nonhydrolyzable myristoyl-CoA analog, S-(2-oxopentadecyl)-CoA, blocked the GTP effect identically to myristoyl- and palmitoyl-CoA (IC50 = 0.5 microM); thus, fatty acyl transfer is not required, indicating that blockade is due to a direct allosteric modification of a component of the GTP-activated process by acyl-CoA esters. Palmitoyl-CoA not only inhibited but completely reversed GTP-activated Ca2+ release, resulting in the released Ca2+ being taken back up into pools. In the presence of oxalate, GTP-activated Ca2+ transfer results in a substantial increase in Ca2+ accumulation; palmitoyl-CoA also completely reversed this effect resulting in rapid termination of Ca2+ uptake. This reversal provides strong evidence that GTP-activated Ca2+ translocation does not reflect a membrane fusion event. Instead, it likely represents formation of a reversible junction or pore between organelles which may be a required prefusion event.

Acylation↗
PubMed1994

The role of calcium, pH, and cell proliferation in the programmed (apoptotic) death of androgen-independent prostatic cancer cells induced by thapsigargin.

Calcium (Ca2+) accumulates within the endoplasmic reticulum of cells through function of the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase family of intracellular Ca(2+)-pumping ATPases. The resulting pools have important signaling functions. Thapsigargin (TG) is a sesquiterpene gamma-lactone which selectively inhibits the sarcoplasmic reticulum and endoplasmic reticulum Ca(2+)-dependent ATPase pumps with a 50% inhibitory concentration of approximately 30 nM. Treatment of androgen-independent prostate cancer cells of both rat and human origin with TG inhibits their endoplasmic reticulum Ca(2+)-dependent ATPase activity, resulting in a 3-4-fold elevation in the level of intracellular free Ca2+ (Cai) within minutes of exposure. Due to a secondary influx of extracellular Ca2+, this increase in Cai is sustained, resulting in morphological (cell rounding) and biochemical changes within 6-12 h (enhanced calmodulin, glucose regulated protein, and tissue transglutaminase expression, and decreased expression of the G1 cyclins). Within 24 h of exposure, androgen-independent prostatic cancer cells stop progression through the cell cycle, arrest out of cycle in G0, and irreversibly lose their ability to proliferate with a median effective concentration value of 31 nM TG. During the next 24-48 h, the genomic DNA of the G0-arrested cells undergoes double-strand fragmentation. This is followed by the loss of plasma membrane integrity and fragmentation of the cell into apoptotic bodies. During this process, there is no acidification in the intracellular pH. Using cells transfected with the avian M(r) 28,000 calbindin D Ca(2+)-buffering protein, it was demonstrated that the programmed death initiated by TG is critically dependent upon an adequate (i.e., 3-4-fold) sustained (> 1 h) elevation in Cai and not depletion of the endoplasmic reticulum pools of Ca2+. These results demonstrate that TG induces programmed cell death in androgen-independent prostatic cancer cells in a dose-dependent manner and that this death does not require proliferation or intracellular acidification but is critically dependent upon an adequate, sustained (i.e., > 1 h) elevation in Cai.

Androgens↗
PubMed1994

Sphingosine 1-phosphate generated in the endoplasmic reticulum membrane activates release of stored calcium.

Sphingosine and sphingosine derivatives induce Ca2+ release from inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools in permeabilized cells (Ghosh, T. K., Bian, J., and Gill, D. L. (1990) Science 248, 1653-1656). To further assess the mechanism of sphingoid base-mediated Ca2+ release, the effects of sphingosine and sphingosine derivatives on Ca2+ fluxes were characterized using a microsomal membrane vesicle fraction (B3) enriched in rough endoplasmic reticulum (ER) prepared from cells of the DDT1MF-2 cell smooth muscle line (Ghosh, T. K., Mullaney, J. M., Tarazi, F. I., and Gill, D. L. (1989) Nature 340, 236-239). Addition of 15 microM sphingosine to Ca2+ pump-loaded B3 vesicles induced a delayed but thereafter rapid Ca2+ release from vesicles which was dependent on the presence of ATP and was blocked by ADP. Sphingosylphosphorylcholine (SPC) released Ca2+ to the same extent (more than 80% of pumped Ca2+), but in contrast to sphingosine, there was no lag and the effect was independent of ATP or ADP. The EC50 for sphingosine and SPC in activating Ca2+ release was 1 and 3 microM, respectively. Such observations are consistent with the view that sphingosine, unlike SPC, must be modified by an ATP-requiring kinase activity located within the ER membrane. Sphingoid bases do not appear to release Ca2+ through InsP3 receptors since heparin had no effect on sphingoid base-mediated Ca2+ release. Sphingosine 1-phosphate (sph-1-P), the likely active Ca(2+)-releasing derivative of sphingosine, was synthesized by phospholipase D-catalyzed cleavage of SPC, purified, and tested for Ca(2+)-releasing activity. sph-1-P at 10 microM induced Ca2+ release from both B3 vesicles and permeabilized DDT1MF-2 cells to exactly the same extent as sphingosine. Unlike sphingosine, the effect of sph-1-P was immediate and not blocked by ADP. Using B3 membrane vesicles incubated with [gamma-32P]ATP and sphingosine under the same conditions as Ca2+ flux studies, a labeled band was detected on TLC which ran identically with authentic sph-1-P. Formation of this labeled product was prevented by removal of exogenous sphingosine and blocked by ADP. Sphingosine- but not SPC-mediated Ca2+ release was blocked by 10 mM oxalate. 10 mM oxalate also blocked the formation of 32P-labeled sph-1-P indicating that it is an inhibitor of sph-1-P formation. The studies establish that the ER membrane contains the necessary kinase to convert sphingosine to sph-1-P which functions as a powerful mediator of Ca2+ release through a non-InsP3 receptor-mediated mechanism in the same ER membrane, perhaps reflecting a novel Ca2+ signaling pathway.

Adenosine Diphosphate↗
PubMed1994

Endoplasmic reticulum calcium pump expression and control of cell growth.

Intracellular Ca2+ pump expression and Ca2+ pool function are shown to be closely associated with growth and proliferation of DDT1MF-2 hamster smooth muscle cells. The Ca2+ pump blocker thapsigargin induces sustained Ca2+ pool emptying and entry of cells into a quiescent G0-like state (Short, A. D., Bian, J., Ghosh, T. K., Waldron, R. T., Rybak, S. L., and Gill, D. L. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4986-4990). Using DDT1MF-2 cells growth-arrested by exposure to 3 microM thapsigargin for 24 h, treatment with 20% serum for 6 h without thapsigargin induced expression of functional Ca2+ pump protein detected as a 110-kDa thapsigargin-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. New functional Ca2+ pump protein could be detected within 1 h of high serum treatment of thapsigargin-arrested cells, increasing over a 6-h period and correlating with the appearance of new Ca2+ pools. Induction of Ca2+ pools required serum at 10% or higher; no pools appeared with 5% serum or less. Significantly, high serum was required for only a brief but precise period of time. Exposure of thapsigargin-arrested cells to a 45-min pulse of 20% serum followed by continued culture in 2.5% serum was sufficient for full induction of new functional Ca2+ pump protein and Ca2+ pools; in contrast, no pumps or pools were detected after a 30-min serum pulse. A 40-min high serum pulse resulted in arrested cells reentering the cell cycle, synthesizing DNA, and resuming normal proliferation; in contrast, 35 min of serum treatment resulted in cells remaining totally quiescent. The results provide important evidence for the necessity of functional endoplasmic reticulum Ca2+ pumps in serum-induced cell growth and reflect a remarkably precise signaling period during which quiescent cells become committed to a progression of events including Ca2+ pump expression, Ca2+ pool function, reentry into the cell cycle, and cell division.

Animals↗
PubMed1993

Inositol 1,4,5-trisphosphate-mediated quantal Ca2+ release measured by high resolution imaging of Ca2+ within organelles.

The distribution and operation of Ca2+ pools within cells has been directly studied in situ by monitoring the Ca2+ inside Ca2+ dye-loaded organelles using high resolution imaging procedures. Using DDT1MF-2 smooth muscle cells, loaded with fura-2 under conditions favoring dye entry into organelles and subjected to carefully controlled permeabilization still attached to coverslips, the Ca2+ within organelles was analyzed by high resolution, z axis-controlled imaging, and deblurring methods. Saturation analysis of entrapped fura-2 indicated that the dye reported Ca2+ identically to fura-2 in solution. Areas containing high Ca(2+)-sequestering organelles (> 5 microM free Ca2+) were observed to predominate around the nucleus and close to the periphery of the cell. Analysis of the actions of inositol 1,4,5-trisphosphate (InsP3) within small (3 microns 2) selected intracellular areas, revealed a "quantal" release phenomenon, with rapid attainment of limited stable release at submaximal InsP3 levels. The apparent EC50 for InsP3 was approximately 3 microns, higher than within suspensions of permeabilized cells. The action of InsP3 was competitively blocked by 10 micrograms/ml of the InsP3 antagonist, heparin. Applied after maximal InsP3-mediated Ca2+ release, heparin reversed InsP3-induced Ca2+ release resulting in reuptake of Ca2+ into Ca(2+)-pumping organelles with identical spatial distribution as before Ca2+ release. InsP3 released Ca2+ from all areas of high Ca(2+)-pumping organelles; extensive areas of high fura-2-loading, but low intraorganelle Ca2+, were unchanged by InsP3. GTP induced no alteration in Ca2+ release (in contrast to suspensions of permeabilized cells), suggesting that the InsP3-sensitive Ca2+ pool was functioning as a single homogeneous pool. Opening of InsP3-sensitive channels was also monitored by assessing InsP3-activated channel-mediated Mn2+ quenching of organelle-loaded fura-2; the results revealed a similar pattern of quantal release, with slightly increased apparent InsP3 sensitivity. The results provide the first high resolution in situ localization of Ca2+ signaling organelles and demonstrate the quantal operation of InsP3-sensitive Ca2+ pools within highly discrete subcellular loci.

Animals↗
PubMed1993

Intracellular Ca2+ pool content is linked to control of cell growth.

A close correlation was observed between intracellular Ca2+ pool depletion and refilling and the onset of DNA synthesis and proliferation of DDT1MF-2 smooth muscle cells. The intracellular Ca2+ pump inhibitors 2,5-di-tert-butyl-hydroquinone (DBHQ) and thapsigargin (TG) specifically emptied identical inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pools and both arrested cell growth at concentrations corresponding to Ca2+ pump blockade. However, an important distinction was observed between the two inhibitors with respect to their reversibility of action. Upon removal of DBHQ from DBHQ-arrested cells, Ca2+ pools immediately refilled, and 14 hr later cells entered S phase followed by normal cell proliferation; the time for entry into S phase was identical to that for cells released from confluence arrest. Although TG irreversibly blocked Ca2+ pumping and emptied Ca2+ pools, high serum treatment of TG-arrested cells induced recovery of functional Ca2+ pools in 6 hr (via probable synthesis of new pump); thereafter cells proceeded to S phase and normal cell proliferation within the same time period (14 hr) as that following release of DBHQ-arrested cells. The precise relationship between Ca2+ pump blockade and growth arrest indicates that Ca2+ pool emptying maintains cells in a G0-like quiescent state; upon refilling of pools, normal progression into the cell cycle is resumed. It is possible that a specific cell cycle event necessary for G0 to G1 transition depends upon signals generated from the InsP3-sensitive Ca2+ pool.

Animals↗
PubMed1993

An examination of personal/situational variables, stress appraisal, and burnout in collegiate teacher-coaches.

Following Smith's (1986) cognitive-affective model of stress and burnout in athletics, this investigation examined (a) the relationship of personal/situational variables (social support, gender, and years of experience) to stress appraisal and (b) the relationship of stress appraisal (perceived stress, coaching issues, and role conflict) to burnout. Male (n = 99) and female (n = 115) teacher-head basketball coaches from NCAA Division III and NAIA colleges completed established measures of burnout, perceived stress, teacher-coach role conflict, and social support and a measure of coaching issues developed for this study. Multivariate analyses supported the hypothesized relationships. Specifically, greater satisfaction with social support, less experience, and gender (females higher) were related to stress appraisal, and all stress appraisal variables were positively related to burnout. Contrary to previous studies, these teacher-coaches reported moderate to high levels of burnout.

Burnout, Professional↗
PubMed1992

Strengthening physical self-perceptions through exercise.

Two studies examined the effects of physical activity/exercise on physical self-perceptions, self-efficacy, body satisfaction, fitness and relationships among these variables. In study 1, 34 female undergraduates participated in a 10-week exercise/activity program. Participants were selected from existing classes forming a weight training, aerobic exercise and activity control group. Results revealed changes in physical self-perceptions, strength, and body composition over the 10-weeks. Improvements in physical self-perceptions and fitness occurred independent of exercise/activity group. Groups differed in the perceived importance attached to physical self-perceptions. Correlations among the measures revealed relationships among physical self-perceptions, body satisfaction, global self-esteem, and fitness. In study 2, we hypothesized that weight training would have a greater effect on physical self-perceptions and body image perceptions than physical education activity classes. Thirty-seven males and 28 females were selected from existing classes forming a weight training and activity group. Results revealed no significant changes in physical self-perceptions, body image, or global self-esteem over the 10-week program, while strength and physical self-efficacy improved. Correlations among measures from both studies offer preliminary support for Sonstroem and Morgan's model for the examination of self-esteem in exercise settings.

Adolescent↗
PubMed1991

Persistent intracellular calcium pool depletion by thapsigargin and its influence on cell growth.

The intracellular Ca2+ pump inhibitor, thapsigargin, added to DDT1MF-2 smooth muscle cells in culture, irreversibly inhibited accumulation of Ca2+ within cells, permanently emptied the inositol 1,4,5-trisphosphate (InsP3)-sensitive Ca2+ pool, and simultaneously induced profound alteration of cell growth. After only a brief (30-min) treatment of cultured cells with 3 microM thapsigargin followed by extensive washing, the total releasable InsP3-sensitive Ca2+ pool remained entirely empty, even after 7 days of culture without thapsigargin. After thapsigargin treatment, cells retained viability, usual morphology, and normal mitochondrial function. Despite the otherwise normal appearance and function of thapsigargin-treated cells, cell division was completely blocked by thapsigargin. DNA synthesis was completely inhibited when thapsigargin was added immediately after passaging, but was suppressed only slowly (4-6 h) when added to rapidly synthesizing cells (24 h after passaging). Protein synthesis was reduced by approximately 70% in thapsigargin-treated cells. The sensitivity of thapsigargin-mediated inhibition of cell division, DNA synthesis, protein synthesis, and Ca(2+)-pumping activity were all similar with the EC50 values for thapsigargin in each case being close to 10 nM. Upon application to DDT1MF-2 cells, thapsigargin transiently increased resting cytosolic Ca2+ (0.15 microM) to a peak of 0.3 microM within 50 s; thereafter, free Ca2+ declined to 0.2 microM by 150 s and continued to slowly decline toward resting levels. Cells treated with thapsigargin for 1-72 h in culture displayed normal resting cytosolic Ca2+ levels. However, application of thapsigargin or epinephrine to such cells resulted in no change in the intracellular Ca2+, indicating that the internal Ca2+ pool remained completely empty. These results suggest that emptying of Ca2+ from intracellular thapsigargin-sensitive Ca(2+)-pumping pools induces profound alteration of cell proliferation.

Animals↗
PubMed1991

Identification of intracellular calcium pools. Selective modification by thapsigargin.

Recent studies have identified inositol 1,4,5-tris-phosphate(InsP3)-sensitive and -insensitive Ca2+ pools and a GTP-dependent mechanism that transfers Ca2+ between them. Here, the Ca2+ pump-inhibitory sesquiterpene lactone, thapsigargin, is shown to distinguish these two Ca2+ pools and identify a third Ca2+ pumping pool unresponsive to InsP3 or GTP. Using saponin-permeabilized DDT1MF-2 smooth muscle cells, approximately 75% of total intracellular ATP-dependent Ca2+ accumulation is blocked by thapsigargin with an IC50 of 30 nM. In contrast, 1 mM vanadate or 5 microM A23187 block 100% of Ca2+ accumulation. The thapsigargin-responsive Ca2+ pool corresponds exactly to that released by 10 microM InsP3 in the presence of 10 microM GTP. Indeed, addition of InsP3 with GTP has no effect on Ca2+ accumulated in the presence of 3 microM thapsigargin whereas A23187 releases all the remaining Ca2+. Added after maximal Ca2+ uptake, thapsigargin induces only slow Ca2+ release consistent with blockade of pumping activity. Unlike InsP3, the action of thapsigargin is entirely heparin insensitive. The large increment in Ca2+ uptake caused by 12 mM oxalate is completely reversed by thapsigargin, indicating that thapsigargin functions on an oxalate-permeable pool. Moreover, the still larger uptake induced by GTP in the presence of oxalate is also completely reversed by either thapsigargin or InsP3. The results indicate that thasigargin blocks Ca2+ uptake into two discrete pools: the InsP3-sensitive, oxalate-permeable Ca2+ pool and the InsP3-insensitive, oxalate-impermeable Ca2+ pool that can be "recruited" into the InsP3-sensitive pool by GTP-dependent Ca2+ translocation (Ghosh, T. K., Mullaney, J.M., Tarazi, F.I., and Gill, D.L. (1989) Nature 340, 236-239). Additionally, a third Ca2+ pool is defined, unreleasable by InsP3 or GTP, and containing a thapsigargin-insensitive Ca2+ pump.

Animals↗
PubMed1990

Intracellular calcium release mediated by sphingosine derivatives generated in cells.

Soluble and hydrophobic lipid breakdown products have a variety of important signaling roles in cells. Here sphingoid bases derived in cells from sphingolipid breakdown are shown to have a potent and direct effect in mediating calcium release from intracellular stores. Sphingosine must be enzymically converted within the cell to a product believed to be sphingosine-1-phosphate, which thereafter effects calcium release from a pool including the inositol 1,4,5-trisphosphate-sensitive calcium pool. The sensitivity, molecular specificity, and reversibility of the effect on calcium movements closely parallel sphingoid base-mediated inhibition of protein kinase C. Generation of sphingoid bases in cells may activate a dual signaling pathway involving regulation of calcium and protein kinase C, comparable perhaps to the phosphatidylinositol and calcium signaling pathway.

Adenosine Diphosphate↗