Inhibitory Effect Of Thapsigargin Research Articles

Calcium and the replication of human vascular smooth muscle cells: studies on the activation and translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression

Since the precise role of sarcoplasmic reticular Ca 2+ in mediating vascular smooth muscle cells (VSMC) proliferation is unknown, the effect of pre-incubation with thapsigargin on extracellular signal regulated kinase (ERK) activation, the translocation of activated of ERK 1/2 to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca 2+ levels were studied. Human saphenous vein VSMCs (hVSMC) were incubated with 10 nM thapsigargin for 24 h followed by stimulation with fetal calf serum and the activation of ERK1/2 and cyclin D1 assessed by western blotting, the intracellular distribution of ERK1/2 using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine and sarcoplasmic reticular Ca 2+ status using FURA-2. Thapsigargin had a marginal effect on ERK1/2 activation only at 5 min and 10 min after stimulation with fetal calf serum. In contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by thapsigargin. S phase was delayed by 8 h by thapsigargin which co-incided with the recovery of cytosolic Ca 2+ levels and cyclin D1 expression. It is concluded that the inhibitory effect of thapsigargin (depletion of Ca 2+ pools) on hVSMC replication is mediated through the inhibition of translocation of activated ERK1/2 to the nucleus and not to the phosphorylation of ERK, per se, which in turn prevents cyclin D1 expression and thus progression of the cell cycle.

Effects of Aldosterone and Mineralocorticoid Receptor Blockade on Intracellular Electrolytes

Genomic mechanisms of mineralocorticoid action have been increasingly elucidated over the past four decades. In renal epithelia, the main effect is an increase in sodium transport through activation and de novo synthesis of epithelial sodium channels. This leads to increased concentrations of intracellular sodium activating sodium-potassium-ATPase molecules mainly at the basolateral membrane which extrude sodium back into the blood stream. In contrast, rapid steroid actions have been widely recognized only recently. The present article summarizes both traditional and rapid effects of mineralocorticoid hormones on intracellular electrolytes, e.g. free intracellular calcium in vascular smooth muscle cells as determined by fura 2 spectrofluorometry in single cultured cells from rat aorta. Latter effects are almost immediate, reach a plateau after only 3 to 5 minutes and are characterized by high specificity for mineralocorticoids versus glucocorticoids. The effect of aldosterone is blocked by neomycin and short-term treatment with phorbol esters but augmented by staurosporine, indicating an involvement of phospholipase C and protein kinase C. The Ca(2+) effect appears to involve the release of intracellular Ca(2+), as shown by the inhibitory effect of thapsigargin. This mechanism operates at physiological subnanomolar aldosterone concentrations and appears to result in rapid fine tuning of cardiovascular responsivity. As a landmark feature of these rapid effects, insensitivity to classic antimineralocorticosteroids, e.g. spironolactone or canrenone has been found in the majority of observations. In an integrated view, mineralocorticoids seem to mainly effect intracellular electrolytes genomically to induce transepithelial transport, and induce nongenomically mediated alterations of cell function (e.g. vasoconstriction) by rapid effects on intracellular electrolytes such as free intracellular calcium.

An Early Growth Response Protein (Egr) 1 cis-Element Is Required for Gonadotropin-releasing Hormone-induced Mitogen-activated Protein Kinase Phosphatase 2 Gene Expression

In pituitary gonadotropes, gonadotropin-releasing hormone (GnRH) activates all three major mitogen-activated protein kinase (MAPK) cascades. The MAPKs play key roles in transcriptional activation of GnRH-responsive genes. MAPK phosphatases (MKPs) are dual specificity protein phosphatases involved in feedback regulation of MAPK activity. Previous studies indicate that GnRH activates MKP-2 expression in gonadotropes, dependent upon activation of multiple MAPKs and discrete Ca(2+) signals. To further understand the transcriptional mechanism(s) of MKP-2 induction by GnRH, we studied the activity of a 198-nucleotide MKP-2 proximal promoter region that supports GnRH responsiveness in reporter gene assays. Functional analysis of the MKP-2 promoter confirmed a requirement for the protein kinase C-extracellular signal-regulated kinase (ERK) pathway and VGCC-derived Ca(2+) signals in transcriptional activation of the MKP-2 gene. However, the inhibitory effect of thapsigargin on MKP-2 protein expression previously identified was not mediated at the level of promoter activation, suggesting a distinct mechanism for the action of thapsigargin-sensitive Ca(2+) signals. MGRE (MKP-2 GnRH response element) within the MKP-2 promoter mediated promoter activation through the protein kinase C-ERK pathway. The zinc finger transcription factor Egr-1 was identified in the MGRE-binding complex. Egr-1/MGRE binding was induced by GnRH in an ERK-dependent manner. Transcriptional activity of Egr-1 protein was enhanced by GnRH treatment. In addition, overexpression of the Egr-interacting protein, NAB1, resulted in increased GnRH-stimulated MKP-2 gene transcription. Consistent with the putative role of Egr-1 in MKP-2 promoter regulation, Egr-1 protein expression closely correlated with the expression of MKP-2 protein in alpha T3-1 cells. Together, these data suggest that Egr-1 may be a key factor in mediating GnRH-dependent transcriptional activation of the MKP-2 gene.

Influence of stimulation on Ca 2+ recruitment triggering [ 3H]acetylcholine release from the rat motor-nerve endings

The influence of rat phrenic nerve stimulation frequency (5–50 Hz) and of pulse duration (0.04–1 ms) on Ca 2+ mobilization triggering [ 3H]acetylcholine release was investigated. The P-type voltage-dependent Ca 2+ channel (VDCC) blocker, ω-agatoxin IVA (100 nM), decreased [ 3H]acetylcholine release evoked by pulses of 0.04-ms duration delivered at 5 Hz frequency. When the stimulus pulse duration was increased to 1 ms (5 Hz frequency) or the stimulation frequency to 50 Hz (0.04-ms duration), inhibition of [ 3H]acetylcholine release became evident after blockade of L-type VDCC, with nifedipine (1 μM), and/or depletion of thapsigargin-sensitive internal stores. The inhibitory effect of thapsigargin (2 μM) was still observed in Ca 2+-free medium. Neither ω-conotoxin GVIA (1 μM) nor ω-conotoxin MVIIC (150 nM) modified neurotransmitter release. The results suggest that, depending on the stimulus paradigm, both internal (thapsigargin-sensitive) and external (either P- or L-type channels) Ca 2+ pools can be mobilized to promote acetylcholine release from motor nerve terminals.

Differential regulation of mammalian brain-specific proline transporter by calcium and calcium-dependent protein kinases.

1. This study examined the role of [Ca2+]I and Ca(2+)-dependent kinases in the modulation of high-affinity, mammalian brain-specific L-proline transporter (PROT). 2. beta-PMA (phorbol 12-myristate 13-acetate), an activator of protein kinase C (PKC), inhibits PRO uptake, and bisindolymalemide I (BIM), a potent PKC inhibitor, prevents beta-PMA inhibition. Down-regulation of PKC by chronic treatment with beta-PMA enhances PROT function indicating PROT regulation by tonic activity of PKC. 3. Thapsigargin, which increases [Ca2+]I levels by inhibiting Ca(2+)-ATPase, inhibits PROT and exhibits additive inhibition when co-treated with beta-PMA. KN-62, a Ca2+/calmodulin-dependent kinase II (CaMK II) inhibitor, but not BIM (a PKC inhibitor) prevents the inhibition by thapsigargin. These data suggest that PKC and CaMK II modulate PROT and that thapsigargin mediates its effect via CaMK II. 4. Thapsigargin raises [Ca2+]I and increases PRO-induced current on a second time scale, whereas the inhibitory effect of thapsigargin occurs only after 10 min of treatment. These data suggest that Ca2+ differentially regulate PROT: Ca2+ initially enhances PRO transport but eventually inhibits transport function through CaMK II pathway. 5. Ca(2+)-induced stimulation exemplifies the acute regulation of a neurotransmitter transporter, which may play a critical role in the profile of neurotransmitters during synaptic transmission.

Intracrine role of progesterone in fibronectin production and deposition by chicken ovarian granulosa cells in vitro: effect of extracellular calcium.

The role of extracellular Ca2+ in progesterone-induced fibronectin production and deposition by hen granulosa cells was studied. Granulosa cells isolated from preovulatory follicles of the chicken ovary were incubated in Ca(2+)-deficient or Ca(2+)-containing medium 199, and the amount of total fibronectin produced (fibronectin deposited in the matrix, secreted into the medium, or associated with cells) was measured by ELISA. The quantity of fibronectin deposited as well as the total amount of fibronectin produced in the presence or absence of exogenous progesterone was suppressed in Ca(2+)-deficient medium. It required greater concentrations of exogenous progesterone to significantly increase fibronectin production in Ca(2+)-deficient medium than in Ca(2+)-replete medium. Cyanoketone, an inhibitor of progesterone synthesis, suppressed total fibronectin produced by unstimulated cells both in the absence and presence of Ca2+. However, the inhibitory effect of cyanoketone was significantly less in Ca(2+)-replete medium than in Ca(2+)-deficient medium. Exogenous progesterone reversed completely the inhibitory effects of cyanoketone. In the presence of cyanoketone, progesterone caused a greater (3-fold) increase in fibronectin production in Ca(2+)-replete medium than in the absence of Ca2+. Thapsigargin, an agent that mobilizes Ca2+ from internal stores, suppressed basal and progesterone-induced fibronectin production in the absence and presence of cyanoketone. However, the inhibitory effect of thapsigargin was significantly reduced in Ca(2+)-replete medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two different Ca2+ uptake and IP3-sensitive Ca2+ release mechanisms in microsomal Ca2+ pools of rat pancreatic acinar cells

We have examined the effect of the Ca2+ (Mg2+)-ATPase inhibitors thapsigargin (TG) and vanadate on ATP-dependent 45Ca2+ uptake into IP3-sensitive Ca2+ pools in isolated microsomes from rat pancreatic acinar cells. The inhibitory effect of TG was biphasic. About 40-50% of total Ca2+ uptake was inhibited by TG up to 10 nM (apparent Ki approximately 4.2 nM, Ca2+ pool I). An additional increase of inhibition up to 85-90% of total Ca2+ uptake could be achieved at 15 to 20 nM of TG (apparent Ki approximately 12.1 nM, Ca2+ pool II). The rest was due to TG-insensitive contaminating plasma membranes and could be inhibited by vanadate (apparent Ki approximately 10 microM). In the absence of TG, increasing concentrations of vanadate also showed two phases of inhibition of microsomal Ca2+ uptake. About 30-40% of total Ca2+ uptake was inhibited by 100 microM of vanadate (apparent Ki approximately 18 microM, Ca2+ pool II). The remaining 60-70% could be inhibited either by vanadate at concentrations up to 1 mM (apparent Ki approximately 300 microM) or by TG up to 10 nM (Ca2+ pool I). The amount of IP3-induced Ca2+ release was constant at approximately 25% over a wide range of Ca2+ filling. About 10-20% remained unreleasable by IP3. Reduction of IP3-releasable Ca2+ in the presence of inhibitors showed similar dose-response curves as Ca2+ uptake (apparent Ki approximately 3.0 nM for IP3-induced Ca2+ release as compared to approximately 4.2 nM for Ca2+ uptake at TG up to 10 nM) indicating that the highly TG-sensitive Ca2+ pump fills the IP3-sensitive Ca2+ pool I. At TG concentrations > 10 nM which blocked Ca2+ pool II the apparent Ki values were approximately 11.3 and approximately 12.1 nM, respectively. For inhibition by vanadate up to 100 microM the apparent Ki values were approximately 18 microM for Ca2+ uptake and approximately 7 microM for Ca2+ release (Ca2+ pool II). At vanadate concentrations up to 1 mM the apparent Ki values were approximately 300 and approximately 200 microM, respectively (Ca2+ pool I). Both Ca2+ pools I and II also showed different sensitivities to IP3. Dose-response curves for IP3 in the absence of inhibitors (control) showed an apparent Km value for IP3 at 0.6 microM. In the presence of TG (inhibition of Ca2+ pool I) the curve was shifted to the left with an apparent Km for IP3 at 0.08 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Thapsigargin inhibits voltage-activated calcium channels in adrenal glomerulosa cells

Thapsigargin, an inhibitor of the microsomal Ca2+ pumps, has been extensively used to study the intracellular Ca2+ pool participating in the generation of the agonist-induced Ca2+ signal in various cell types. A dual effect of this agent was observed in bovine adrenal zona glomerulosa cells. At nanomolar concentrations, thapsigargin stimulated a sustained Ca2+ influx, probably resulting from Ca(2+)-store depletion. In contrast, when added at micromolar concentrations, thapsigargin prevented the rise in cytosolic free Ca2+ concentration ([Ca2+]c) induced by K+. This inhibitory effect of thapsigargin on voltage-activated Ca2+ channels was confirmed by measuring Ba2+ currents by the patch-clamp technique. Both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels were affected by micromolar concentrations of thapsigargin. Analysis of the current-voltage relationship for T-type channels revealed that thapsigargin did not modify the sensitivity of these channels to the voltage, but decreased the maximal current flowing through the channels. In conclusion, thapsigargin appears to exert a dual effect on adrenal glomerulosa cells. At lower concentrations, this agent induces a sustained Ca2+ entry, whereas at higher concentrations it decreases [Ca2+]c by blocking voltage-activated Ca2+ channels.

Inhibition of phosphatidylserine synthesis by glutamate, acetylcholine, thapsigargin and ionophore A23187 in glioma C6 cells

Phosphatidylserine synthesis was studied in glioma C6 cells with [14C]serine and in the presence or absence of agents which increase the level of [Ca2+]i. It was found that glutamate and acetylcholine inhibited this synthesis by up to 40%, whereas thapsigargin and the ionophore A23187 inhibited by up to 70%. The inhibitory effect of thapsigargin and the A23187 was observed in Ca(2+)-free medium. The data show that the inhibition of this synthesis is caused by the Ca(2+)-depletion from endoplasmic reticulum, suggesting that the synthesis of phosphatidylserine occurs on the luminal side of these structures and can be regulated by transmembrane signaling systems.

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.