thapsigargin and 1-oleoyl-2-acetylglycerol

Although ethanol is known to inhibit platelet aggregation, the effects of another variant of alcohol, methanol, have not been reported. The purpose of this study was to determine whether methanol and its metabolite, formic acid, affect Ca. Thrombin-induced platelet aggregation was significantly augmented by methanol at pharmacological concentrations (0.5-2%) in a concentration-dependent manner. Methanol at 2% significantly attenuated thapsigargin-induced platelet aggregation, which was not significantly affected by lower concentrations (0.5 and 1%) of methanol. Methanol (0.5-2%) did not significantly affect platelet aggregation induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or Ca. Methanol at pharmacological doses has diverse effects on platelet aggregation, depending on the aggregation stimuli, without affecting Ca

Topics: Blood Platelets; Calcium; Calcium Signaling; Diglycerides; Formates; Hemostatics; Humans; Methanol; Platelet Aggregation; Thapsigargin; Thrombin

Expression of transient receptor potential canonical channels (TRPC) and the effects of transforming growth factor-β1 (TGF-β1) on Ca2+ signals and fibroblast proliferation were investigated in human cardiac fibroblasts. The conventional and quantitative real-time RT-PCR, western blot, immunocytochemical analysis, and intracellular Ca2+ concentration [Ca2+]i measurement were applied. Cell proliferation and cell cycle progression were assessed using MTT assays and fluorescence activated cell sorting. Human cardiac fibroblasts have the expression of TRPC1,3,4,6 mRNA and proteins. 1-oleoyl-2-acetyl-sn-glycerol (OAG) and thapsigargin induced extracellular Ca(2+)-mediated [Ca2+]i rise. siRNA for knock down of TRPC6 reduced OAG-induced Ca2+ entry. Hyperforin as well as angiotensin II (Ang II) induced Ca2+ entry. KB-R7943, a reverse-mode Na+/Ca2+ exchanger (NCX) inhibitor, and/or replacement of Na+ with NMDG+ inhibited thapsigargin-, OAG- and Ang II-induced Ca2+ entry. Treatment with TGF-β1 increased thapsigargin-, OAG- and Ang II-induced Ca2+ entry with an enhancement of TRPC1,6 protein expression, suppressed by KB-R7943. TGF-β1 and AngII promoted cell cycle progression from G0/G1 to S/G2/M and cell proliferation. A decrease of the extracellular Ca2+ and KB-R7943 suppressed it. Human cardiac fibroblasts contain several TRPC-mediated Ca2+ influx pathways, which activate the reverse-mode NCX. TGF-β1 enhances the Ca2+ influx pathways requiring Ca2+ signals for its effect on fibroblast proliferation.

Topics: Angiotensin II; Calcium; Calcium Signaling; Cell Cycle Checkpoints; Cell Line; Cell Proliferation; Diglycerides; Enzyme Inhibitors; Fibroblasts; Humans; Phloroglucinol; RNA Interference; RNA, Messenger; RNA, Small Interfering; Sodium-Calcium Exchanger; Terpenes; Thapsigargin; Thiourea; Transforming Growth Factor beta1; TRPC Cation Channels

The requirement of calcium ion (Ca(2)(+)) entry for neutrophil NADPH oxidase (NOX2) regulation is clearly established. However, its role in the signaling pathway leading to NOX2 activation is still elusive. 1-oleoyl-2-acetyl-sn-glycerol (OAG) causes an increase in NOX2 activity and has been shown to directly modulate Ca(2)(+) channels unrelated to the well-known store-operated Ca(2)(+) entry (SOCE) mechanism. In our study, we have investigated the potential role of OAG in Ca(2)(+) influx-mediated NOX2 activity in neutrophil-like-differentiated HL-60 cells to further characterize second signals involved in the regulation of NOX2. OAG inhibited fMLF- and thapsigargin-induced Ca(2)(+) entry, a phenomenon that was not restored by protein kinase C (PKC) or PI3K blockade. Addition of OAG resulted in a rapid decrease of maximal intracellular Ca(2)(+) concentration induced by thapsigargin. Both results suggest that OAG has an inhibitory effect, independent of PI3K and PKC, on the regulation of SOCE. In contrast to SOCE inhibition, OAG-induced NOX2 activation was mediated by PKC and PI3K. Our data establish that both kinases exert their effects through the regulation of Rac2 activity. In addition, OAG potentiated the effect of fMLF on the activation of NOX2 and led to a discernible activity of NOX2 upon thapsigargin stimulation. In conclusion, our results demonstrate that an additional PKC- and/or PI3K-dependent signal may act in synergy with Ca(2)(+) influx to trigger NOX2 activation.

Topics: Calcium; Diglycerides; HL-60 Cells; Humans; Membrane Glycoproteins; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidase 2; NADPH Oxidases; Phosphatidylinositol 3-Kinases; Protein Kinase C; rac GTP-Binding Proteins; RAC2 GTP-Binding Protein; Signal Transduction; Thapsigargin; Up-Regulation

Repetitive hormone-induced changes in concentration of free cytoplasmic Ca2+ in hepatocytes require Ca2+ entry through receptor-activated Ca2+ channels and SOCs (store-operated Ca2+ channels). SOCs are activated by a decrease in Ca2+ concentration in the intracellular Ca2+ stores, but the molecular components and mechanisms are not well understood. Some studies with other cell types suggest that PLC-gamma (phospholipase C-gamma) is involved in the activation of receptor-activated Ca2+ channels and/or SOCs, independently of PLC-gamma-mediated generation of IP3 (inositol 1,4,5-trisphosphate). The nature of the Ca2+ channels regulated by PLC-gamma has not been defined clearly. The aim of the present study was to determine if PLC-gamma is required for the activation of SOCs in liver cells. Transfection of H4IIE cells derived from rat hepatocytes with siRNA (short interfering RNA) targeted to PLC-gamma1 caused a reduction (by approx. 70%) in the PLC-gamma1 protein expression, with maximal effect at 72-96 h. This was associated with a decrease (by approx. 60%) in the amplitude of the I(SOC) (store-operated Ca2+ current) developed in response to intracellular perfusion with either IP(3) or thapsigargin. Knockdown of STIM1 (stromal interaction molecule type 1) by siRNA also resulted in a significant reduction (approx. 80% at 72 h post-transfection) of the I(SOC) amplitude. Immunoprecipitation of PLC-gamma1 and STIM1, however, suggested that under the experimental conditions these proteins do not interact with each other. It is concluded that the PLC-gamma1 protein, independently of IP3 generation and STIM1, is required to couple endoplasmic reticulum Ca2+ release to the activation of SOCs in the plasma membrane of H4IIE liver cells.

Topics: Animals; Calcium; Calcium Channels; Cells, Cultured; Diglycerides; Estrenes; Hepatocytes; Inositol 1,4,5-Trisphosphate; Membrane Glycoproteins; Phosphatidylinositol 4,5-Diphosphate; Phospholipase C gamma; Pyrrolidinones; Rats; RNA, Small Interfering; Stromal Interaction Molecule 1; Thapsigargin; Transfection

We recently showed that increased expression of the transient receptor potential canonical Type 3 (TRPC3) channel is associated with genetic hypertension. It is unknown whether store-operated TRPC3 channels, which are activated after depletion of intracellular stores, or second messenger-operated TRPC3 channels, which are activated by 1-oleoyl-2-acetyl-sn-glycerol, show augmented responses in monocytes in genetic hypertension and support the development of vascular disease.. Using the fluorescent-dye technique, we studied store-depleted and thapsigargin-induced, store-operated calcium influx and 1-oleoyl-2-acetyl-sn-glycerol-induced second messenger-operated calcium influx into monocytes from spontaneously hypertensive rats (SHRs) and from normotensive Wistar-Kyoto rats (WKYs). The RNA interference for the downregulation of TRPC3 in monocytes by small, interfering RNA (siRNA) was performed and evaluated using in-cell Western assay.. Thapsigargin-induced, store-operated calcium influx was significantly elevated in SHRs and was approximately double that observed in WKYs. In the presence of nimodipine, the thapsigargin-induced, store-operated calcium influx was also significantly higher in SHRs compared with WKYs. After stimulation of monocytes by angiotensin II, calcium influx was significantly elevated in SHRs, and was approximately double that observed in WKYs. The 1-oleoyl-2-acetyl-sn-glycerol-induced, second messenger-operated calcium influx was also significantly elevated in SHRs compared with WKYs. Thapsigargin-induced, store-operated calcium influx was reduced by the inhibitor 2-aminoethoxydiphenyl borane. After TRPC3 knockdown, the thapsigargin-induced, store-operated calcium influx, as well as 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx, was significantly more reduced in cells from SHRs compared with WKYs.. The increased store-operated and second messenger-operated calcium influx through TRPC3 channels in monocytes from SHRs may be responsible for a more aggressive effect in promoting vascular disease in genetic hypertension.

Topics: Animals; Calcium; Diglycerides; Down-Regulation; Enzyme Inhibitors; Hypertension; Male; Monocytes; Rats; Rats, Inbred SHR; Rats, Inbred WKY; RNA, Small Interfering; Second Messenger Systems; Thapsigargin; TRPC Cation Channels

Numerous studies have demonstrated that members of the transient receptor potential (TRP) superfamily of channels are involved in regulated Ca2+ entry. Additionally, most Ca2+-permeable channels are themselves regulated by Ca2+, often in complex ways. In the current study, we have investigated the regulation of TRPC7, a channel known to be potentially activated by both store-operated mechanisms and non-store-operated mechanisms involving diacylglycerols. Surprisingly, we found that activation of TRPC7 channels by diacylglycerol was blocked by the SERCA pump inhibitor thapsigargin. The structurally related channel, TRPC3, was similarly inhibited. This effect depended on extracellular calcium and on the driving force for Ca2+ entry. The inhibition is not due to calcium entry through store-operated channels but rather results from calcium entry through TRPC7 channels themselves. The effect of thapsigargin was prevented by inhibition of calmodulin and was mimicked by pharmacological disruption of the actin cytoskeleton. Our results suggest the presence of a novel mechanism involving negative regulation of TRPC channels by calcium entering through the channels. Under physiological conditions, this negative feedback by calcium is attenuated by the presence of closely associated SERCA pumps.

Topics: Adenosine Triphosphate; Boron Compounds; Calcium; Calcium-Transporting ATPases; Calmodulin; Cations; Cytochalasin B; Cytoskeleton; Depsipeptides; Diglycerides; Gadolinium; Humans; Imidazoles; Indoles; Ion Transport; Thapsigargin; TRPC Cation Channels

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca(2+) (Ca(2+))(i) in endothelial cells, resulting in Ca(2+)-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca(2+) influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca(2+) influx was resolved into capacitative Ca(2+) entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 microM) and a phospholipase C (PLC) inhibitor (U73122; 10 microM). The action of bradykinin could be mimicked by 1-oleoyl-2-acetyl-glycerol, an analogue of diacylglycerol (DAG), and by RHC80267, a DAG-lipase inhibitor. Immunoblots showed that TRPC6 was one of the main TRPC channels expressed in endothelial cells. Transfection of H5V cells with two siRNA constructs against TRPC6 both markedly reduced the TRPC6 protein level and, at the same time, decreased the percentage of cells displaying bradykinin-induced non-CCE. siRNA transfection also reduced the magnitude of non-CCE among the cells responding to bradykinin. Taken together, our data suggest that bradykinin-induced non-CCE is mediated via the B2-PLC pathway, and that DAG may be involved in this process. Further, TRPC6 is one of the important channels participating in bradykinin-induced non-CCE in endothelial cells.

Topics: Animals; Bradykinin; Calcium; Calcium-Transporting ATPases; Cell Line, Transformed; Diglycerides; Endothelial Cells; Enzyme Inhibitors; Estrenes; Male; Mice; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; RNA Interference; RNA, Small Interfering; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Signal Transduction; Thapsigargin; Time Factors; Transfection; TRPC Cation Channels; TRPC6 Cation Channel; Type C Phospholipases; Vasodilator Agents

Altered 1-oleoyl-lysophosphatidic acid (LPA, 100 microM)-stimulated calcium responses occur in B-lymphoblast cell lines from bipolar disorder patients, but the mechanism(s) involved is uncertain. Lysophosphatidic acid shares a structurally similar fatty acid side chain with the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a known activator of subtypes 3, 6 and 7 of the canonical transient receptor potential (TRPC) cation channel subfamily. Accordingly, the objective of this study was to determine whether the LPA-stimulated calcium response in B-lymphoblasts is mediated, in part, through this TRPC channel subfamily. Divalent cation selectivity in response to thapsigargin, LPA and OAG were used to distinguish TRPC-like character of the responses to these agents in BLCLs. The sensitivity to gadolinium, an inhibitor of capacitative calcium channels, was used to determine the store-operated nature of the responses. The TRPC isoforms that are present in BLCLs as identified by immunoblotting and/or PCR include TRPC1, 3 and 5. Minimal barium influx in calcium-free buffer was observed following thapsigargin stimulation. However, LPA stimulated barium influx of a magnitude similar to that induced by OAG. Thapsigargin-provoked calcium influx was completely inhibited by gadolinium (10 microM), whereas LPA and OAG-stimulated responses were partially inhibited and potentiated, respectively. The results suggest that 100 microM LPA stimulates calcium entry through channels with characteristics similar to TRPC3, as TRPC6 and 7 are absent in B-lymphoblasts.

Topics: B-Lymphocytes; Barium; Bipolar Disorder; Calcium Signaling; Cell Line; Diglycerides; Humans; Immunoblotting; Lysophospholipids; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Thapsigargin; TRPC Cation Channels

We have reported that internal Ca2+ store depletion in HSY cells stimulates a nonselective cation current which is distinct from I(CRAC) in RBL cells and TRPC1-dependent I(SOC) in HSG cells (Liu, X., Groschner, K., and Ambudkar, I. S. (2004) J. Membr. Biol. 200, 93-104). Here we have analyzed the molecular composition of this channel. Both thapsigargin (Tg) and 2-acetyl-sn-glycerol (OAG) stimulated similar non-selective cation currents and Ca2+ entry in HSY cells. The effects of Tg and OAG were not additive. HSY cells endogenously expressed TRPC1, TRPC3, and TRPC4 but not TRPC5 or TRPC6. Immunoprecipitation of TRPC1 pulled down TRPC3 but not TRPC4. Conversely, TRPC1 co-immunoprecipitated with TRPC3. Expression of antisense TRPC1 decreased (i) Tg- and OAG-stimulated currents and Ca2+ entry and (ii) the level of endogenous TRPC1 but not TRPC4. Antisense TRPC3 similarly reduced Ca2+ entry and endogenous TRPC3. Yeast two-hybrid analysis revealed an interaction between NTRPC1 and NTRPC3 (CTRPC1-CTRPC3, CTRPC3-CTRPC1, or CTRPC1-NTRPC3 did not interact), which was confirmed by glutathione S-transferase (GST) pull-down assays (GST-NTRPC3 pulled down TRPC1 and vice versa). Expression of NTRPC1 or NTRPC3 induced similar dominant suppression of Tg- and OAG-stimulated Ca2+ entry. NTRPC3 did not alter surface expression of TRPC1 or TRPC3 but disrupted TRPC1-TRPC3 association. In aggregate, our data demonstrate that TRPC1 and TRPC3 co-assemble, via N-terminal interactions, to form a heteromeric store-operated non-selective cation channel in HSY cells. Thus selective association between TRPCs generate distinct store-operated channels. Diversity of store-operated channels might be related to the physiology of the different cell types.

Topics: beta-Galactosidase; Biotinylation; Blotting, Western; Calcium; Calcium Channels; Cations; Cell Line; Cell Membrane; Diglycerides; Electrophysiology; Genes, Dominant; Glutathione Transferase; Humans; Immunoprecipitation; Ion Channels; Oligonucleotides, Antisense; Patch-Clamp Techniques; Perfusion; Protein Binding; Protein Structure, Tertiary; Thapsigargin; Transfection; TRPC Cation Channels; Two-Hybrid System Techniques

The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bore the hallmarks of TRPC6 function; it was inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry was blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels would allow depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction.

Topics: Animals; Blotting, Western; Calcium; Calcium Channels; Cations; Diglycerides; DNA Primers; Electrophysiology; Electroporation; Fura-2; Ions; Membrane Potentials; Models, Biological; Myocytes, Smooth Muscle; Nimodipine; Oligonucleotides; Patch-Clamp Techniques; Pyrimidines; Rats; Receptors, Vasopressin; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Signal Transduction; Sodium; Thapsigargin; Time Factors; TRPC Cation Channels; TRPC6 Cation Channel; Type C Phospholipases; Vasopressins

Chronic hypoxic pulmonary hypertension is associated with profound vascular remodeling and alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Recent studies show that transient receptor potential (TRPC) genes, which encode store-operated and receptor-operated cation channels, play important roles in Ca(2+) regulation and cell proliferation. However, the influence of chronic hypoxia on TRPC channels has not been determined. Here we compared TRPC expression, and store- and receptor-operated Ca(2+) entries in PASMCs of normoxic and chronic hypoxic rats. Reverse-transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining showed consistently that TRPC1, TRPC3, and TRPC6 were expressed in intralobar pulmonary arteries (PAs) and PASMCs. Application of 1-oleoyl-2-acetyl-sn-glycerol (OAG) to directly activate receptor-operated channels, or thapsigargin to deplete Ca(2+) stores, caused dramatic increase in cation entry measured by Mn(2+) quenching of fura-2 and by Ca(2+) transients. OAG-induced responses were approximately 700-fold more resistant to La(3+) inhibition than thapsigargin-induced responses. siRNA knockdown of TRPC1 and TRPC6 specifically attenuated thapsigargin- and OAG-induced cation entries, respectively, indicating that TRPC1 mediates store-operated entry and TRPC6 mediates receptor-operated entry. In hypoxic PAs, there were 2- to 3-fold increases in TRPC1 and TRPC6 expression. They were accompanied by significant increases in basal, OAG-induced, and thapsigargin-induced cation entries in hypoxic PASMCs. Moreover, removal of Ca(2+) or inhibition of store-operated Ca(2+) entry with La(3+) and SK&F-96365 reversed the elevated basal [Ca(2+)](i) in PASMCs and vascular tone in PAs of chronic hypoxic animals, but nifedipine had minimal effects. Our results for the first time to our knowledge show that both store- and receptor-operated channels of PASMCs are upregulated by chronic hypoxia and contribute to the enhanced vascular tone in hypoxic pulmonary hypertension.

Topics: Animals; Calcium; Calcium Channels; Cations; Cell Hypoxia; Cells, Cultured; Diglycerides; Hypertension, Pulmonary; Ion Transport; Male; Muscle, Smooth, Vascular; Pulmonary Artery; Rats; Rats, Wistar; RNA Interference; Thapsigargin; TRPC Cation Channels; Up-Regulation

Astrocytes express transient receptor potential channels (TRPCs), which have been implicated in Ca 2+ influx triggered by intracellular Ca 2+ stores depletion, a phenomenon known as capacitative Ca 2+ entry. We studied the properties of capacitative Ca 2+ entry in astrocytes by means of single-cell Ca 2+ imaging with the aim of understanding the involvement of TRPCs in this function. We found that, in astrocytes, capacitative Ca 2+ entry is not attributable to TRPC opening because the TRPC-permeable ions Sr2+ and Ba2+ do not enter astrocytes during capacitative Ca 2+ entry. Instead, natively expressed oleyl-acetyl-glycerol (OAG) (a structural analog of DAG) -sensitive TRPCs, when activated, initiate oscillations of cytosolic Ca 2+ concentration ([Ca 2+]i) pharmacologically and molecularly consistent with TRPC3 activation. OAG-induced [Ca 2+]i oscillations are not affected by inhibition of inositol trisphosphate (InsP3) production or blockade of the InsP3 receptor, therefore representing a novel form of [Ca 2+]i signaling. Instead, high [Ca 2+]i inhibited oscillations, by closing the OAG-sensitive channel. Also, treatment of astrocytes with antisense against TRPC3 caused a consistent decrease of the cells responding to OAG. Exogenous OAG but not endogenous DAG seems to activate TRPC3. In conclusion, in glial cells, natively expressed TRPC3s mediates a novel form of Ca 2+ signaling, distinct from capacitative Ca 2+ entry, which suggests a specific signaling function for this channel in glial cells.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Biological Clocks; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Diglycerides; Enzyme Inhibitors; Fluorescent Dyes; Glioma; Immunohistochemistry; Intracellular Fluid; Ion Channels; Neuroglia; Oligonucleotides, Antisense; Protein Isoforms; Rats; Rats, Wistar; Strontium; Thapsigargin; TRPC Cation Channels

Protein kinase C and calmodulin play key roles in cockroach fat body during activation of phosphorylase and trehalose efflux by HTH-II. The data support the view that an increase in cytosolic Ca2+ is prerequisite for enhanced activity of protein kinase C and calmodulin. Chelation of Ca2+ (i) with BAPTA blocks HTH-II-induced trehalose efflux from the fat body whereas thapsigargin, which raises [Ca2+]i to the same level as HTH-II, produces only a small, yet significant increase in trehalose efflux. Sphingosine, an inhibitor of protein kinase C, inhibits HTH-II-induced trehalose efflux in a concentration-dependent manner. Trehalose efflux is not activated by the protein kinase C activators OAG or PMA alone but in the presence of thapsigargin both agents increase trehalose efflux to a level comparable to that obtained with HTH-II. Thapsigargin has only a moderate activating effect on phosphorylase but in combination with OAG produces an activation indistinguishable from that provoked by HTH-II. Each of the structurally different calmodulin inhibitors, trifluoperazine, W-7, and calmidazolium, blocks completely the action of HTH-II on trehalose efflux, thus confirming the importance of calmodulin in HTH-II initiated trehalose efflux.

Topics: Animals; Calcium; Calmodulin; Chelating Agents; Diglycerides; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Fat Body; Male; Neuropeptides; Periplaneta; Phosphorylases; Protein Kinase C; Sphingosine; Tetradecanoylphorbol Acetate; Thapsigargin; Trehalose; Trifluoperazine

In this study, we report the molecular cloning of cDNAs encoding three distinct isoforms of rat (r) TRP6 Ca(2+) channels. The longest isoform, rTRP6A, contains 930 amino acid residues; rTRP6B lacks 54 amino acids (3-56) at the N terminus, and rTRP6C is missing an additional 68 amino acids near the C terminus. Transient transfection of COS cells with expression vectors encoding rTRP6A or rTRP6B increased Ca(2+) influx and gave rise to a novel Ba(2+) influx after activation of M(5) muscarinic acetylcholine receptors. By contrast, passive depletion of intracellular Ca(2+) stores with thapsigargin did not induce Ba(2+) influx in cells expressing rTRP6 isoforms. Ba(2+) influx was also stimulated in rTRP6A-expressing cells after exposure to the diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), but rTRP6B-expressing cells failed to show OAG-induced Ba(2+) influx. Expression of a rTRP6 N-terminal fragment of rTRP6B or rTRP6A antisense RNA blocked M(5) muscarinic acetylcholine receptor-dependent Ba(2+) influx in COS cells that were transfected with rTRP6 cDNAs. Together these results suggest that rTRP6 participates in the formation of Ca(2+) channels that are regulated by a G-protein-coupled receptor, but not by intracellular Ca(2+) stores. In contrast to the results we obtained with rTRP6A and rTRP6B, cells expressing rTRP6C showed no increased Ca(2+) or Ba(2+) influxes after stimulation with carbachol and also did not show OAG-induced Ba(2+) influx. Glycosylation analysis indicated that rTRP6A and rTRP6B are glycosylated in COS cells, but that rTRP6C is mostly not glycosylated. Together these results suggest that the N terminus (3-56 amino acids) is crucial for the activation of rTRP6A by diacylglycerol and that the 735-802 amino acid segment located just downstream from the 6th transmembrane segment may be required for processing of the rTRP6 protein.

Topics: Amino Acid Sequence; Animals; Barium; Calcium; Calcium Channels; Carbachol; Chlorocebus aethiops; Cloning, Molecular; COS Cells; Diglycerides; Molecular Sequence Data; Protein Isoforms; Rats; Receptor, Muscarinic M5; Receptors, Muscarinic; Recombinant Proteins; RNA, Antisense; Sequence Alignment; Sequence Homology, Amino Acid; Thapsigargin; Transfection; TRPC Cation Channels

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.

Topics: Animals; Calcium; Cell Membrane; Diglycerides; Female; Humans; Ion Channels; Ionomycin; Jurkat Cells; Kinetics; Leukemia, Basophilic, Acute; Lymphocytes; Membrane Potentials; Oocytes; Patch-Clamp Techniques; Rats; Saccharomyces cerevisiae; Second Messenger Systems; T-Lymphocytes; Thapsigargin; Tumor Cells, Cultured; Xenopus laevis

Phorbol 12-myristate 13-acetate, a potential stimulator of protein kinase C (PKC), inhibited taurine uptake in rat astrocytes. This effect was mimicked by 1-oleoyl-2-acetyl-sn-glycerol, an endogenous stimulator of PKC, and by r-59949, an inhibitor of diacylglycerol kinase. Maximal inhibition was obtained at microM phorbol 12-myristate 13-acetate (PMA) after 1 h of treatment. This effect was prevented by pretreatment of the cells with chelerythrine, a potent and selective inhibitor of PKC. The transport of beta-alanine, an amino acid that shares the same transporter as taurine, was inhibited to a comparable extent. The effect of PMA was potentiated by cotreatment of the cells with thapsigargin or the Ca2+ ionophore A-23187. However, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N1,N1-tetraacetic acid and verapamil did not prevent the PMA effect. Pretreatment of the cells with calmodulin antagonists W-13 or calmidazolium, prevented the PMA-induced inhibition of taurine uptake. This inhibition was not affected by cycloheximide, actinomycin D, colchicine, or cytochalasin D. The Na(+)-to-Cl(-)-to-taurine coupling ratio was unaffected. Dimethyl amiloride, a selective inhibitor of Na+/H+ antiport, was unable to prevent the effects of PMA. These effects were associated with a decrease in the maximal velocity and an increase in the Michaelis-Menten constant.

Topics: Animals; Astrocytes; Binding, Competitive; Biological Transport; Calcimycin; Calcium; Calcium-Transporting ATPases; Calmodulin; Diglycerides; Enzyme Activation; Kinetics; Protein Kinase C; Rats; Taurine; Tetradecanoylphorbol Acetate; Thapsigargin

1. The effects of caffeine, isoprenaline, dibutyryl cyclic AMP, isobutylmethylxanthine (IBMX), 12-O-tetradecanoylphorbol-13-acetate (TPA) or 1-oleoyl-2-acetylglycerol (OAG), (protein kinase C (PKC) activators), 2-methoxy verapamil (D600), thapsigargin and ryanodine on muscarinic acetylcholine receptor (AChR)-stimulated inositol phospholipid hydrolysis were studied in smooth muscle fragments from the longitudinal layer of the small intestine of the guinea-pig. 2. Incubation of the fragments with the muscarinic agonist, carbachol (CCh) (100 microM) resulted in rapid increases in the levels of all the inositol phosphate isomers with maximal increases in the [3H]-inositol (1,4,5) trisphosphate ([3H]-Ins(1,4,5)P3) isomer occurring 10 s following incubation. 3. The beta-adrenoceptor agonist, isoprenaline (10 microM) and dibutyryl cyclic AMP (10 microM), a membrane permeant analogue of cyclic AMP both reduced the CCh stimulation, but not the basal levels of [3H]-inositol phosphates. This inhibition by dibutyryl cyclic AMP was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. CCh inhibited the isoprenaline-induced increases in the levels of cyclic AMP and this was via a pertussi toxin (PTX)-sensitive G-protein mechanism. 4. TPA (1 microM) and OAG (100 microM) a 1,2-diacylglycerol (DAG) analogue both reduced the CCh-induced increases in [3H]-inositol phosphates levels but neither affected basal values nor the basal levels of cyclic AMP. 5. D600 (10 microM), which blocks voltage-dependent Ca2+ channels, also reduced the CCh-stimulated levels of [3H]-inositol phosphates suggesting that some of the agonist-induced increases are due to a potentiating effect of Ca2+ entering the cell. 6. Caffeine (0.5-30 mM) significantly inhibited both the basal and CCh-induced increases in all the [3H]-inositol phosphate isomers. Its inhibitory action was not due to increases in cyclic AMP since caffeine had no effect on the levels of cyclic AMP at concentrations up to 30 mM. 7. Incubation with thapsigargin (1 microM) and ryanodine (10 microM) had no effect on either basal or CCh-induced inositol phospholipid hydrolysis or cyclic AMP levels. 8. The results indicate a reciprocal inhibition by beta-adrenoceptors and muscarinic AChRs of their effects on cyclic AMP and inositol phosphate levels respectively. Ca2+ entering the cell (but not the action of ryanodine or thapsigargin) potentiates while caffeine inhibits muscarinic AChR-induced rises in inositol phosphate leve

Topics: 1-Methyl-3-isobutylxanthine; Animals; Bucladesine; Caffeine; Calcium Channels; Calcium-Transporting ATPases; Carbachol; Cyclic AMP; Diglycerides; Gallopamil; Guinea Pigs; Hydrolysis; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Intestine, Small; Isoproterenol; Muscle, Smooth; Protein Kinase C; Receptors, Adrenergic, beta; Receptors, Muscarinic; Ryanodine; Stereoisomerism; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin

A series of studies was conducted to evaluate the ability of several second messengers/second messenger systems to stimulate LH secretion from dispersed chicken pituitary cells. [Gln8]-LHRH-(cLHRH) stimulated LH secretion in a dose-dependent fashion; this effect was potentiated in the presence of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and was mimicked by the cAMP analog, 8-bromo-cAMP. These data indicate that the production of cAMP in response to cLHRH can stimulate LH secretion, but do not necessarily provide evidence that such production is prerequisite. The tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), and diacylglycerol analogs, 1-oleoyl-2-acetylglycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), also stimulated LH release; however, only PMA (and not cLHRH or DOG) promoted an accumulation of cAMP. The putative protein kinase C inhibitor, staurosporine, completely blocked LH release stimulated by PMA, but failed to block cLHRH-induced LH secretion. Such results indicate that protein kinase C activation can promote LH secretion, but also suggest that additional second messengers may exist to fully mediate the effects of cLHRH. Both the calcium ionophore, A23187, and the intracellular calcium mobilizing agent, thapsigargin, caused a dose-dependent increase in LH secretion; furthermore, thapsigargin augmented the stimulatory effects of PMA. These data are consistent with a role for calcium in the regulation of LH release, and indicate that the mobilization of intracellular calcium alone can affect such an action.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-Methyl-3-isobutylxanthine; Alkaloids; Animals; Arachidonic Acids; Calcimycin; Calcium; Carcinogens; Chickens; Cyclic AMP; Diglycerides; Dose-Response Relationship, Drug; Epidermal Growth Factor; Gonadotropin-Releasing Hormone; Luteinizing Hormone; Male; Pituitary Gland; Protein Kinase C; Second Messenger Systems; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Transforming Growth Factor alpha