Effects Of Thapsigargin Research Articles

Thapsigargin, an inhibitor of endoplasmic reticulum Ca2+-ATPase, antagonizes cytokeratin assembly of human hepatoma cells induced by teleocidin

Abstract Teleocidin, a phorbol ester-type of tumor promoter markedly enhanced cytokeratin assembly of PLC/PRF/5 hepatoma cells. Thapsigargin, a non-phorbol ester-type tumor promoter which specifically inhibited endoplasmic reticulum Ca 2+ -ATPase, elevated cytosolic calcium and antagonized the cytokeratin assembly induced by teleocidin. A calcium ionophore ionomycin mimicked the effect of thapsigargin, suggesting that elevated cytosolic calcium appeared to be responsible for the antagonistic action of thapsigargin on the cytokeratin assembly.

Regulation of the mitochondrial ATP-Mg/Pi carrier in isolated hepatocytes.

This study investigated the cellular regulation of net adenine nucleotide movements between the cytoplasm and mitochondria in intact cells. Such movements are presumed to occur primarily by ATP-Mg exchange with Pi via the mitochondrial ATP-Mg/Pi carrier. Vasopressin, A23187, and thapsigargin all elevate intracellular free [Ca2+] and all caused dose-dependent increases in the mitochondrial adenine nucleotide content (29, 63, and 39%, respectively). Phorbol 12-myristate 13-acetate had no effect. The effect of vasopressin was abolished when cytoplasmic [ATP] was decreased (by 43%) and [Pi] was increased (3-fold) by addition of carboxyatractyloside. The effect of thapsigargin was abolished by addition of xylulose to deplete cytoplasmic [ATP] (by 50%) and [Pi] (> 4-fold). The results indicate that in intact cells Ca2+ activates the mitochondrial ATP-Mg/Pi carrier to enable changes in the subcellular distribution of adenine nucleotides and that the relative [ATP] and [Pi] gradients govern the direction and magnitude of net adenine nucleotide movements between the cytoplasm and mitochondria.

Use of calcium pump inhibitors in the study of calcium regulation in smooth muscle.

The study of the relative roles of the plasma membrane (PM) and the sarcoplasmic reticulum (SR) Ca2+ pumps in the regulation of internal Ca2+ has been limited in the past due in part to the lack of selective inhibitors for either Ca2+ pump. Recently, three compounds have been discovered which appear to be selective SR Ca2+ pump inhibitors: thapsigargin (TSG), cyclopiazonic acid (CPA), and 2,5-di-(tert-butyl)-1,4-benzohydroquinone. Contractility studies in various smooth muscle tissues have demonstrated that all three compounds inhibit repletion of the intracellular Ca2+ store, presumably due to inhibition of the SR Ca2+ pump. These functional studies however provided only indirect evidence for Ca2+ pump inhibition. Using the microsomal membrane fraction isolated from the smooth muscle of rat vas deferens, we investigated the effects of CPA and TSG of ATP-dependent Ca2+ uptake in order to obtain direct evidence about the mechanism of action of CPA and TSG on smooth muscle Ca2+ pumps. CPA and TSG potently inhibited oxalate-stimulated Ca2+ uptake in a concentration-dependent manner. Since oxalate-stimulated Ca2+ uptake is generally considered to be a property of the SR and not the PM Ca2+ pump, this directly demonstrates that CPA and TSG act by inhibiting the SR Ca2+ pump. We also demonstrated that inhibition of Ca2+ uptake in the presence of oxalate by CPA could be completely reversed while the effects of TSG could only be partially reversed.(ABSTRACT TRUNCATED AT 250 WORDS)

The Effect of Thapsigargin on Sarcoplasmic Reticulum Ca 2+ Content and Contractions in Single Myocytes of Guinea-pig Heart

Contractures initiated by 1 s superfusion of single myocytes of guinea-pig heart with 10 mM caffeine were used as a relative index of the SR Ca 2+ content. Thapsigargin (Tg) in concentration 2 × 10 -7M completely blocked Ca 2+ uptake during electrical stimulation by the SR from which Ca 2+ has been previously depleted by caffeine. Tg did not affect the SR Ca 2+ content in the resting myocytes and did not block release of the SR Ca 2+ during electrically stimulated contractions (ESCs). It is concluded that in guinea-pig myocytes Tg affects SR Ca 2+ by selective blocking the SR Ca 2+ uptake. The amplitude of steady state ESCs dropped to 68±5.4% (S.D., n=20) of that of the pre-Tg control. Time to peak contraction increased from 454±82.4 ms to 820±157.4 ms and time of relaxation increased from 368±90.8 ms to 474±87 ms after the SR Ca 2+ has been depleted by Tg. Rest decay of contractions, post-extrasystolic potentiation and post-rest potentiation were inhibited.

Effects of thapsigargin and ryanodine on vascular contractility: cross-talk between sacroplasmic reticulum and plasmalemma

Thapsigargin and ryanodine are proposed to interfere with Ca 2+ storage in sarcoplasmic reticulum by different mechanisms. Thapsigargin inhibits Ca 2+ transport into and ryanodine enhances Ca 2+ out of the sarcoplasmic reticulum. Contractility studies were performed in the rat aorta and dog mesenteric artery. Ryanodine was found to reduce phenylephrine-induced (10 μM) contraction in Ca 2+-free medium of rat aorta and dog mesenteric artery in a concentration-dependent manner. Each agent alone caused a slow contraction in the rat aorta. In this tissue, the tension caused by ryanodine (30 μM) but not that by thapsigargin (1 μM) was found to be dependent on the status of the sarcoplasmic reticulum: prior stimulation with K + (60 mM) enhanced the rate of development of ryanodine-induced tension compared with when the sarcoplasmic reticulum was previously depleted with phenylephrine stimulation in Ca 2+-free medium. Sodium nitroprusside (1 μM) or isoproterenol (1 μM) fully antagonized the contraction induced by ryanodine or phenylephrine. However, thapsigargin-induced contraction was antagonized fully by sodium nitroprusside and only partially by isoproterenol. This result suggests that cAMP elevation by isoproterenol required a functioning sarcoplasmic reticulum Ca 2+ pump for its relaxant effect while cAMP elevation by sodium nitroprusside did not. These findings are consistent with the view that ryanodine promotes Ca 2+ release from the sarcoplasmic reticulum and that thapsigargin inhibits the ability of cAMP to stimulate Ca 2+ uptake into the store by blocking its Ca 2+ pump. In the dog mesenteric artery, when the phenylephrine-sensitive Ca 2+ pool was emptied and thapsigargin was added to block Ca 2+ uptake into the store, restoration of Ca 2+ in the Ca 2+-free medium caused a transient contraction (absent in controls). This contraction was replaced by a significantly larger amplitude and more sustained contraction in low Na + medium indicating the involvement of the Na +/Ca 2+ exchanger in the homeostasis of cytosolic [Ca 2+]. In the presence of nifedipine (2 μM), repletion of the phenylephrine-sensitive store was inhibited. It is possible that refilling occurs in part through L-type Ca 2+ channels.

Effect of thapsigargin and caffeine on Ca2+ homeostasis in HeLa cells: implications for histamine-induced Ca2+ oscillations.

Several studies have already established that the stimulation of H1 receptors by exogenous histamine induces intracellular Ca2+ oscillations in HeLa cells. The molecular mechanism underlying this oscillatory process remains, however, unclear. A series of fura-2 experiments was undertaken in which the nature of the Ca2+ pools involved in the histamine-induced Ca2+ oscillations was investigated using the tumour promoter agent thapsigargin (TG) and the Ca(2+)-induced Ca(2+)-release promoter, caffeine. The results obtained indicate first that TG causes a gradual increase in cytosolic Ca2+ without inducing internal Ca2+ oscillations, and second that TG and histamine share common internal Ca2+ storage sites. The latter conclusion was derived from experiments performed in the absence of external Ca2+, where the addition of TG before histamine resulted in a total inhibition of the Ca2+ response linked to H1 receptor stimulation, whereas the addition of histamine before TG decreased by more than 90% the TG-induced Ca2+ release. Finally; TG was found to inhibit irreversibly histamine-induced Ca2+ oscillations when added to the bathing medium during the oscillatory process. The effect of caffeine at concentrations ranging from 1 mM to 10 mM on intracellular Ca2+ homeostasis was also investigated. The results obtained show that caffeine does not affect systematically the internal Ca2+ concentration in resting and TG-stimulated HeLa cells, but increases the Ca2+ sequestration ability of inositol-trisphosphate (InsP3)-related Ca2+ stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraction without sarcoplasmic reticulum Ca2+: Effect of thapsigargin on single myocytes of guinea-pig and rat heart

Contraction without sarcoplasmic reticulum Ca2+: Effect of thapsigargin on single myocytes of guinea-pig and rat heart

Characterization of the inhibition of intracellular Ca2+ transport ATPases by thapsigargin.

The effects of thapsigargin (TG), a specific inhibitor of intracellular Ca(2+)-ATPases, were studied on vesicular fragments of sarcoplasmic reticulum (SR) membranes. Inhibition of Ca2+ transport and ATPase activity was observed following stoichiometric titration of the membrane bound enzyme with TG. When Ca2+ binding to the enzyme was measured in the absence of ATP, or when one cycle of Ca(2+)-dependent enzyme phosphorylation by ATP was measured under conditions preventing turnover, protection against TG by Ca2+ was observed. The protection by Ca2+ disappeared if the phosphoenzyme was allowed to undergo turnover, indicating that a state reactive to TG is produced during enzyme turnover, whereby a dead end complex with TG is formed. Enzyme phosphorylation with Pi, ATP synthesis, and Ca2+ efflux by the ATPase in its reverse cycling were also inhibited by TG. However, under selected conditions (millimolar Ca2+ in the lumen of the vesicles, and 20% dimethyl sulfoxide in the medium) TG permitted very low rates of enzyme phosphorylation with Pi and ATP synthesis in the presence of ADP. It is concluded that the mechanism of ATPase inhibition by TG involves mutual exclusion of TG and high affinity binding of external Ca2+, as well as strong (but not total) inhibition of other partial reactions of the ATPase cycle. TG reacts selectively with the state acquired by the ATPase in the absence of Ca2+. This state is obtained either by enzyme exposure to EGTA, or by utilization of ATP and consequent displacement of bound Ca2+ during catalytic turnover.

Effect of thapsigargin on cardiac muscle cells

The effect of thapsigargin on the activity of various enzymes involved in the Ca 2+-homeostasis of cardiac muscle and on the contractile activity of isolated cardiomyocytes was investigated. Thapsigargin was found to be a potent and specific inhibitor of the Ca 2+-pump of striated muscle SR (IC 50 in the low nanomolar range). A strong reduction of the V max of the Ca 2+-pump was observed while the Km (Ca 2+) was only slightly affected. Reduction of the V max was caused by the inability of the ATPase to form the Ca 2+-dependent acylphosphate intermediate. Thapsigargin did not change the passive permeability characteristics nor the function of the Ca 2+-release channels of the cisternal compartments of the SR. In addition, no significant effects of thapsigargin on other ATPases, such as the Ca 2+-ATPase and the Na + K +-ATPase of the plasma membrane as well as the actomyosin ATPase could be detected. The contractile activity of paced adult rat cardiomyocytes was completely abolished by 300 nM thapsigargin. At lower concentrations the drug prolonged considerably the contraction-relaxation cycle, in particular the relaxation phase. The intracellular Ca 2+-transients elicited by electrical stimulation (as measured by the changes in Fluo-3 fluorescence) decreased in parallel and the time needed to lower free Ca 2+ down to the resting level increased. In conclusion, the results indicate that selective inhibition of the Ca 2+-pump of the SR by thapsigargin accounts for the functional degeneration of myocytes treated with the drug.

Trypanosoma brucei: The tumor promoter thapsigargin stimulates calcium release from an intracellular compartment in slender bloodstream forms

Maintenance of calcium homeostasis is a critical activity of eukaryotic cells. Homeostatic pathways stabilize intracellular free calcium concentrations ([Ca 2+] i) at the resting level and provide the source of mobilized calcium for cellular activation. We have measured calcium release from intracellular pools within bloodstream forms of Trypanosoma brucei to better understand homeostatic pathways which operate in these organisms. Fura-2 and 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to quantitate [Ca 2+] i and intracellular pH (pH i), respectively. We report that the tumor promoter, thapsigargin, elevated [Ca 2+] i by 50–75 n M. Mn 2+ quench experiments demonstrated that the source of calcium was intracellular. No change in pH i was associated with the release of calcium from this compartment. In contrast, nigericin released approximately three-fold more calcium than thapsigargin from a pH-sensitive, intracellular pool. The nigericin-sensitive pool was nonmitochondrial. The effects of thapsigargin and nigericin on [Ca 2+] i were additive, regardless of the order in which the treatment was given. We conclude that at least two pools of exchangeable calcium occur in bloodstream forms of T. brucei. One pool is sensitive to thapsigargin and apparently resides within the endoplasmic reticulum, while the nigericin-sensitive pool is nonmitochondrial and is of unknown origin.

Thapsigargin increases cytoplasmic free Ca 2+ without influencing steroidogenesis in chicken granulosa cells

The effects of thapsigargin on intracellular Ca 2+ concentration ([Ca 2+] i) and progesterone production were determined in granulosa cells from the two largest preovulatory follicles of laying hens. [Ca 2+] i was measured in cells loaded with the Ca 2+-responsive fluorescent dye Fura-2. Thapsigargin stimulated a 4.6 ± 0.2-fold increase in [Ca 2+] i from a resting level of 55 ± 6 nM up to 233 ± 23 nM (n = 8) in 100% of the cells tested (n = 86). However, two different response patterns were observed. Dependent on the cell populations, a maximally effective concentration of thapsigargin (100 nM) stimulated either a rapid (within 16 ± 2 s) transient increase in [Ca 2+] i or a slowly (99 ± 20 s) developing and sustained increase in [Ca 2+] i. Both [Ca 2+] i responses were concentration (0.001–1 μM)-dependent with an EC 50 around 40 nM. The transient [Ca 2+] i response occurred in the absence of extracellular Ca 2+ and was unaffected by pretreating the cells with the Ca 2+ channel blockers methoxyverapamil (50 μM) or lanthanum (1 mM). The plateau phase of the sustained [Ca 2+] i response returned to resting level in the absence of extracellular Ca 2+, but remained elevated in the presence of methoxyverapamil (50 μM) or lanthanum (1 mM). Despite its ability to cause transient or prolonged increases in [Ca 2+] i, thapsigargin (0.001–1 μM) did not affect basal or luteinizing hormone-stimulated progesterone production by chicken granulosa cells.

Calcium oscillations in parotid acinar cells induced by microsomal Ca(2+)-ATPase inhibition.

Previous studies have demonstrated in single rat parotid acinar cells that the microsomal Ca(2+)-ATPase inhibitor thapsigargin mobilizes Ca2+ specifically from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, activates plasma membrane Ca2+ permeability, and induces intracellular Ca2+ concentration ([Ca2+]i) oscillations that are quite similar to those activated by carbachol. Nevertheless, the IP3-sensitive Ca2+ store remains continuously depleted during thapsigargin-induced oscillations, indicating that this pool is not involved in the oscillation mechanism. To determine the specificity of thapsigargin's effects, in the present study we have examined the effects on [Ca2+]i in single rat parotid acinar cells of two other microsomal Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ), and compared them with the effects of thapsigargin in the same cells. Our results demonstrate that thapsigargin, CPA, and BHQ all similarly deplete the IP3-sensitive Ca2+ store specifically, activate plasma membrane Ca2+ influx, and induce [Ca2+]i oscillations, strongly suggesting that these agents have a specific inhibitory action on microsomal Ca(2+)-ATPase activity. BHQ, in addition, inhibits plasma membrane Ca2+ influx. The data lend strong support to a model in which the state of Ca2+ filling of the IP3-sensitive store regulates plasma membrane Ca2+ influx. These results suggest either that a Ca2+ pump is involved which is insensitive to structurally dissimilar inhibitors or that a Ca2+ pump is not involved in refilling of the Ca2+ pool involved in [Ca2+]i oscillations in these cells.

The effects of thapsigargin on [Ca2+]i in isolated rat mesenteric artery vascular smooth muscle cells.

The effects of thapsigargin were studied on single cells isolated from side branches of the rat mesenteric artery. Thapsigargin (150 nM) produced a transient increase of [Ca2+]i. This transient rise of [Ca2+]i was unaffected by removing external Ca2+ ions. This suggests that thapsigargin is releasing Ca2+ ions from an intracellular store. In the absence of thapsigargin both noradrenaline and caffeine also produced a transient increase of [Ca2+]i. These increases were abolished by prior exposure to thapsigargin. Correspondingly, the effects of thapsigargin were abolished by prior exposure to caffeine. These results show that thapsigargin releases Ca2+ from the noradrenaline and caffeine-sensitive stores.

Biochemical and Ultrastructural Studies Suggest that the Effects of Thapsigargin on Human Platelets Are Mediated by Changes in Intracellular Calcium but not by Intracellular Histamine

The involvement of intracellular histamine in thapsigargin (Tg)-induced platelet aggregation was studied. Platelet aggregation induced by 0.25 and 0.5 microM Tg was not accompanied by a rise in intracellular histamine but a significant (p < 0.01) increase in the level of intracellular histamine was observed at 1 microM Tg. Preincubation of platelets with inhibitors of histamine metabolizing enzymes had little effect on intracellular histamine levels in platelets stimulated by 0.5 microM Tg. In addition, the inhibitors of histidine decarboxylase (HDC), alpha-methyl histidine (alpha-MH) and alpha-fluoromethyl histidine (alpha-FMH) failed to inhibit Tg-induced aggregation. The intracellular histamine receptor antagonist, N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine. HCl (DPPE), inhibited Tg-induced aggregation but with IC50 values dependent on the concentration of agonist used. The inhibitory effects of DPPE on Tg-induced aggregation were not reversed by the addition of histamine to saponin-permeabilized platelets suggesting non-histamine mediated effects of DPPE on Tg-induced aggregation. Tg stimulated an increase in the cytosolic free calcium concentration which was unaffected by DPPE indicating that the effects of DPPE are also not due to the inhibition of mobilization of cytosolic calcium. The ultrastructural studies suggest that the major Tg-induced changes (pseudopod formation and granule centralization) are consistent with a primary role for Tg to mobilize calcium; DPPE had very little effect on these ultrastructural changes. The results indicate that the effects of Tg on human platelets are mediated by an increase in cytosolic calcium but not by intracellular histamine.

Coupling between intracellular Ca2+ stores and the Ca2+ permeability of the plasma membrane. Comparison of the effects of thapsigargin, 2,5-di-(tert-butyl)-1,4-hydroquinone, and cyclopiazonic acid in rat thymic lymphocytes.

The regulation of Ca2+ uptake by receptors is incompletely understood. It has been proposed that the Ca2+ permeability of the plasma membrane increases in response to depletion of a critical intracellular Ca2+ storage compartment (Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W. (1989) J. Biol. Chem. 264, 12266-12271). This hypothesis is based largely on the effect of thapsigargin, an inhibitor of endomembrane CA(2+)-ATPases. Due to the existence of an endogenous leak, inhibition of Ca2+ uptake by thapsigargin induces depletion of the stores. This is accompanied by increased plasmalemmal Ca2+ permeability, without change in the level of inositol phosphates. On the other hand, depletion of the intracellular stores by 2,5-di(tert-butyl)-1,4-hydroquinone (BHQ), a chemically unrelated inhibitor of the Ca(2+)-ATPases, fails to induce Ca2+ influx (Kass, G. E., Duddy, S. K., Moore, G. A., and Orrenius, S. (1989) J. Biol. Chem. 264, 15192-15198). In an attempt to reconcile these observations, we analyzed in lymphocytes the mode of action of thapsigargin and BHQ. In addition, we tested the effects of cyclopiazonic acid (CPA), a blocker of the skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase. All three compounds released Ca2+ from a common intracellular compartment. Thapsigargin and low concentrations of BHQ and CPA concomitantly elevated the plasmalemmal Ca2+ permeability. Higher concentrations of BHQ and CPA produced a secondary inhibition of the Ca2+ entry pathway, by a mechanism seemingly unrelated to their effects on the internal stores. This inhibitory side effect can account for the reported discrepancies between the effects of thapsigargin and BHQ. The data provide further support for the notion that endomembrane Ca2+ stores are functionally coupled to the plasma membrane Ca2+ permeability pathway.

Calcium and Calmodulin Mediation of Growth Hormone-Releasing Hormone-Release from the Rat Hypothalamusin Vitro

A major role for Ca2+ and calmodulin in stimulus-secretion coupling has been suggested for several neuropeptides; however, the cellular mechanisms of GH-releasing hormone (GHRH) release have been little investigated so far. We have used a previously validated acute rat hypothalamic explant system in order to elucidate whether Ca2+ acts as a second messenger in the regulation of GHRH release, and whether calmodulin-dependent pathways are involved. Calcium dependence of somatostatin (SRIH) release was assessed in the same experiments. Calmodulin dependence of SRIH was not investigated in detail, as it has been established previously. The calcium-entry antagonist, verapamil, antagonized K(+)-stimulated GHRH and SRIH release in a dose-dependent manner, with maximal inhibition shown at 10(-4) M. The calmodulin antagonist W7 also blocked K(+)-evoked GHRH release in a dose-dependent manner, with significant inhibition in the dose range 5 X 10(-5) M to 2 X 10(-4) M; similarly, a more specific calmodulin inhibitor, the W7 derivative 5-iodo-C8 (W8), reversed K(+)-stimulated GHRH release, showing slightly higher potency than W7. W7 also reversed GHRH release in response to the calcium-ionophore A23187, although verapamil had no effect on A23187-evoked GHRH or SRIH release. Thapsigargin, which increases the efflux of Ca2+ from calciosomes, did not affect either GHRH or SRIH release at 10(-5) M or 10(-4) M. The basal release of GHRH was clearly suppressed by W7 and W8 (10(-4) M), whereas verapamil had no effect. We conclude that calcium influx is crucial for depolarization-induced GHRH and SRIH release. Calcium entrance in response to A23187 appears to be independent of verapamil-sensitive calcium channels. The lack of effect of thapsigargin suggests that increased intracellular Ca2+ from intracellular stores is not equivalent to an increase in Ca2+ influx. Both basal and depolarization-induced release of GHRH in this system are calmodulin dependent.

Thapsigargin demonstrates calcium-dependent regulation of phosphate uptake in HeLa cells

We used thapsigargin, a sesquiterpene lactone that mobilizes intracellular Ca without increases in inositol phosphates or major activation of protein kinase C (PKC), to test the specific effects of increasing cytosolic Ca on Na-dependent phosphate uptake in HeLa cells. Thapsigargin increased the Vmax for phosphate uptake from 5.40 +/- 0.26 to 7.86 +/- 0.43 nmol.mg protein-1.3 min-1 (n = 7, P less than 0.001) without change in the apparent Km for phosphate, which averaged 0.15 +/- 0.02 mM. The effect of thapsigargin was dependent on concentration and time. Inactivation of PKC by overnight exposure to 16 microM phorbol 12,13-dibutyrate did not eliminate the effect of thapsigargin, although it completely abolished the effects of phorbol ester on phosphate uptake. Thus thapsigargin are not dependent on PKC. As in other cell systems, thapsigargin increased cytosolic Ca concentration. Removal of extracellular Ca diminished the increase in cytosolic Ca and eliminated the effect of thapsigargin on phosphate uptake. Collectively, our data indicate that Na-dependent phosphate uptake in HeLa cells can be regulated by at least three specific signaling pathways: protein kinase A, PKC, and increased cytosolic Ca.

The Calcium-Mobilizing Agent, Thapsigargin, Inhibits Progesterone Production in Rat Luteal Cells by a Calcium-Independent Mechanism*

In rat luteal cells, an increase in intracellular [Ca]i impairs luteal function similar to that of prostaglandin F2a (PGF2a). However, calcium per se is not the mediator of the antigonadotropic action of PGF2a. Thapsigargin, a plant sesquiterpene lactone, increases intracellular calcium concentration concentration ([Ca]i) in several cell types by a mechanism that involves specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase. To further investigate the antigonadotropic role of [Ca]i and the mechanism of action of PGF2a in rat luteal cells, the action of thapsigargin on cellular functional responses was examined in the absence and presence of PGF2a. Thapsigargin dose dependently increased [Ca]i and inhibited cAMP accumulation and progesterone production in response to LH. The inhibitory effect of thapsigargin on cAMP accumulation was calcium dependent but in contrast, inhibition of LH-stimulated progesterone production was independent of calcium mobilization by thapsigargin. Steroidogenesis stimulated by (Bu)2cAMP was also inhibited by thapsigargin. Thus, thapsigargin mimicked some effects of PGF2a with inhibitory sites of action on both cAMP accumulation and progesterone production. Thapsigargin also blocked the mobilization of [Ca]i by PGF2a, but when coincubated with PGF2a an additive effect on inhibition of LH-stimulated progesterone production occurred. However, no additive effects of thapsigargin and PGF2a on gonadotropin-sensitive cAMP accumulation were evident. In conclusion, although thapsigargin and PGF2a may share some similar actions, their antigonadotropic effects are mediated differently.

Effect of thapsigargin on cytoplasmic Ca 2+ and proliferation of human lymphocytes in relations to AIDS

The tumuour-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca 2+ concentrations ([Ca 2+] i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 μM. Half-maximum response was found at about 1 nM of thapsigargin, full response at 100 nM. The effect of thapsigargin on [Ca 2+], expected that of phytohaemagglutinin (PHA) which raised [Ca 2+] i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas the thapsigargin /PMA treatment could not restore the defective proliferation of AIDS lymphocytes in spite of the increased [Ca 2+] i. Thapsigargin or PMA added separately had no stimulatory effects on cell profileration. The thapsigargin/PMA treatment caused an increase in interleukin-2 (IL-2) production of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects on lymphocyte proliferation by increasing [Ca 2+] i, and that the general defect of AIDS lymphocytes, rather than being ascribed to the initiating signal systems, is associated with later events related to DNA synthesis and proliferation.

Effect of thapsigargin on cytoplasmic Ca 2+ and proliferation of human lymphocytes in relation to AIDS

Effect of thapsigargin on cytoplasmic Ca 2+ and proliferation of human lymphocytes in relation to AIDS