Synergistic actions of diacylglycerol and inositol 1,4,5 trisphosphate for Ca^2＋-dependent inactivation of TRPC7 channel

AIM: The aim of the present study was to explore the mechanism for the Ca2+- dependent inactivation of the canonical transient receptor potential (TRPC) 7 channel expressed in human embryonic kidney 293 cells. METHOD: The whole-cell patch-clamp technique was used in the study. RESULTS: With Ca2+-free external solution, the perfusion of 100 micromol/L carbachol to, or dialysis of the cell with 100 micromol/L guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), induced large inward currents, respectively. These currents were rapidly inhibited by the addition of 1 mmol/L Ca2+ into the bath, and recovery from this inhibition was only partial after the Ca2+ removal, unless vigorous intracellular Ca2+ buffering with 10 mmol/L 1,2 bis(2- aminophenoxy)ethane-N,N,No,No-tetraacetic acid (BAPTA) (plus 4 mmol/L Ca2+) was employed. In contrast, the current induced by a membrane-permeable analog of diacylglycerol (DAG), 1-oleoyl-2-acetyl-sn-glycerol (OAG; 100 micromol/L) did not undergo the inhibition persisting after Ca2+ removal. Interestingly, the inclusion of inositol 1,4,5 trisphosphate (IP3; 100 micromol/L) in the patch pipette rendered the OAG-induced current susceptible to the persistent Ca2+-mediated inhibition independent of the IP3 receptor in the majority of the tested cells, as evidenced by the inability of heparin and thapsigargin in reversing the effect of IP3. CONCLUSION: The present results suggest that Ca2+ entry via the activated TRPC7 channel plays a critical role in inactivating the channel where the cooperative actions of DAG and IP3 are essentially involved.     

Function and regulation of endothelin type A receptor-operated transient receptor potential canonical channels

The purpose of this study is to identify transient receptor potential canonical (TRPC) channels responsible for receptor-operated Ca(2+) entry (ROCE) triggered by activation of endothelin type A receptor (ET(A)R) and to clarify the importance of calmodulin (CaM) / inositol 1,4,5-trisphosphate (IP(3)) receptor binding (CIRB) domain at the C terminus of TRPC channels in ET(A)R-activated channel regulation. In HEK293 cells coexpressing ET(A)R and one of seven TRPC isoforms, ET(A)R stimulation induced ROCE through TRPC3, TRPC5, TRPC6, and TRPC7. The TRPC3- and TRPC6-mediated ROCE was inhibited by selective inhibitors of G(q) protein, phospholipase C (PLC), and CaM. The CIRB domain deletion mutants of TRPC3 and TRPC6 failed to induce ET(A)R-mediated ROCE. Either deletion of the CIRB domain or pharmacological inhibition of CaM did not inhibit the targeting of these channels to the plasma membrane. These results suggest that 1) TRPC3, TRPC5, TRPC6, and TRPC7 can function as ET(A)R-operated Ca(2+) channels; 2) G(q) protein, PLC, and CaM are involved in TRPC3- and TRPC6-mediated ROCE; 3) ET(A)R-mediated activation of TRPC3 and TRPC6 requires the CIRB domain; and 4) abolition of ET(A)R-induced ROCE by CIRB domain deletion and CaM inhibition is due to loss of CaM binding to the channels but not loss of cell surface TRPC3 and TRPC6.     

Pharmacological profile of store-operated Ca(2+) entry in intrapulmonary artery smooth muscle cells

Store-operated Ca(2+) entry (SOCE) plays an important role in the contraction and proliferation of pulmonary artery smooth muscle cells (PASMCs). The aim of this study was to characterise the pharmacological properties of the SOCE pathway in freshly isolated PASMCs from rat lung and to determine whether this Ca(2+) entry pathway is sensitive to nitric oxide donor drugs. Following depletion of Ca(2+) from the sarcoplasmic reticulum, by treating cells with thapsigargin, re-addition of Ca(2+) produced an increase in cytosolic fluo-4 fluorescence that was sustained for the period that extracellular Ca(2+) was present. Thapsigargin also increased the rate of quench of fura-2 fluorescence, confirming that SOCE was activated. The SOCE pathway was not affected by nifedipine or verapamil; however, it was inhibited by the divalent cations Ni(2+) (10 microM) and Cd(2+) (10 microM) by 47+/-5% and 49+/-5% respectively. SOCE was also inhibited 42+/-5% by 2-aminoethoxydiphenyl borate (2-APB; 75 microM) and 58+/-4% by Gd(3+) (10 microM), although La(3+) (100 microM) had little effect. None of the NO donors examined, including sodium nitroprusside, glyceryl trinitrate, and 2-(N,N-diethylamino)-diazenolate-2-oxide had any effect on SOCE. Thus, the pulmonary vasorelaxation produced by NO does not involve direct inhibition of SOCE in PASMCs. Western blot and immunocytochemistry using antibodies directed against specific TRPC subunits detected the presence of TRPC1, 3, and 6 in pulmonary artery and the pharmacological profile of SOCE in PASMCs favours a role for TRPC1 in mediating the underlying channels that are activated by store depletion.     

Insulin inhibits rat hippocampal neurones via activation of ATP-sensitive K+ and large conductance Ca2+-activated K+ channels

In this study, we have used a combination of immunocytochemical and Ca(2+) imaging techniques to determine the functional localisation of insulin receptors as well as the potential role for insulin in modulating hippocampal synaptic activity. Comparison of insulin receptor and MAP2 labelling demonstrated extensive insulin receptor immunoreactivity on the soma and dendrites of cultured hippocampal neurones. Dual labelling with synapsin 1 also showed punctate insulin receptor labelling associated with synapses. In functional studies, insulin inhibited spontaneous Ca(2+) oscillations evoked in cultured hippocampal neurones following Mg(2+) removal. This action of insulin was mimicked by the ATP-sensitive K(+) (K(ATP)) channel opener diazoxide or the large conductance Ca(2+)-activated K(+) (BK) channel activator NS-1619. Furthermore, application of the K(ATP) channel blocker glybenclamide or the BK channel inhibitors iberiotoxin or charybdotoxin attenuated the actions of insulin, whereas prior incubation with a combination of glybenclamide and iberiotoxin completely blocked insulin action. The ability of insulin to modulate the Ca(2+) oscillations was reduced by the inhibitors of MAPK activation PD 98059 and U0126, but not by the PI 3-kinase inhibitors LY 294002 or wortmannin, indicating that a MAPK-driven process underlies insulin action. In conclusion, insulin inhibits spontaneous Ca(2+) oscillations via a process involving MAPK-driven activation of BK and K(ATP) channels. This process may be a useful therapeutic target for the treatment of epilepsy and certain neurodegenerative diseases.     

Molecular mechanisms for the activation of Ca2+-permeable nonselective cation channels by endothelin-1 in C6 glioma cells

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. It is possible to discriminate between these channels by using the Ca(2+) channel blockers SK&F 96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) and LOE 908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide]. LOE 908 is a blocker for NSCC-1 and NSCC-2, whereas SK&F 96365 is an inhibitor for NSCC-2. The purpose of the present study was to identify the G-proteins that are involved in ET-1-activated Ca(2+) channels in C6 glioma cells. ET-1 activated only NSCC-1 in C6 glioma cells preincubated with U73122 (1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C (PLC) inhibitor. Microinjection of the dominant negative mutant of G(12)/G(13) (G(12)G228A/G(13)G225A) abolished activation of NSCC-1 and NSCC-2. In contrast, pertussis toxin did not affect any of the Ca(2+) channels in the ET-1-stimulated C6 glioma cells. These results indicate that G(12)/G(13) may couple with endothelin receptors and play an important role in the activation of NSCCs in C6 glioma cells. Moreover, the activation mechanisms of NSCC-1 and NSCC-2 by ET-1 were different. NSCC-1 activation depended upon a G(12)/G(13)-dependent cascade, whereas NSCC-2 activation depended upon both G(q)/PLC- and G(12)/G(13)-dependent cascades.     

Role of basal extracellular Ca2+ entry during 5-HT-induced vasoconstriction of canine pulmonary arteries

1. Measurements of artery contraction, cytosolic [Ca(2+)], and Ca(2+) permeability were made to examine contractile and cytosolic [Ca(2+)] responses of canine pulmonary arteries and isolated cells to 5-hydroxytryptamine (5-HT), and to determine the roles of intracellular Ca(2+) release and extracellular Ca(2+) entry in 5-HT responses. 2. The EC(50) for 5-HT-mediated contractions and cytosolic [Ca(2+)] increases was approximately 10(-7) M and responses were inhibited by ketanserin, a 5-HT(2A)-receptor antagonist. 3. 5-HT induced cytosolic [Ca(2+)] increases were blocked by 20 microM Xestospongin-C and by 2-APB (IC(50)=32 microM inhibitors of InsP(3) receptor activation. 4. 5-HT-mediated contractions were reliant on release of InsP(3) but not ryanodine-sensitive Ca(2+) stores. 5. 5-HT-mediated contractions and cytosolic [Ca(2+)] increases were partially inhibited by 10 microM nisoldipine, a voltage-dependent Ca(2+) channel blocker. 6. Extracellular Ca(2+) removal reduced 5-HT-mediated contractions further than nisoldipine and ablated cytosolic [Ca(2+)] increases and [Ca(2+)] oscillations. Similar to Ca(2+) removal, Ni(2+) reduced cytosolic [Ca(2+)] and [Ca(2+)] oscillations. 7. Mn(2+) quench of fura-2 and voltage-clamp experiments showed that 5-HT failed to activate any significant voltage-independent Ca(2+) entry pathways, including store-operated and receptor-activated nonselective cation channels. Ni(2+) but not nisoldipine or Gd(3+) blocked basal Mn(2+) entry. 8. Voltage-clamp experiments showed that simultaneous depletion of both InsP(3) and ryanodine-sensitive intracellular Ca(2+) stores activates a current with linear voltage dependence and a reversal potential consistent with it being a nonselective cation channel. 5-HT did not activate this current. 9. Basal Ca(2+) entry, rather than CCE, is important to maintain 5-HT-induced cytosolic [Ca(2+)] responses and contraction in canine pulmonary artery.     

Capacitative and 1-oleyl-2-acetyl-sn-glycerol-activated Ca(2+) entry distinguished using adenylyl cyclase type 8

Although the molecular identity of capacitative Ca(2+) entry (CCE) channels remains elusive, transient receptor potential channel (TRPC) family members 3, 6, and 7, which are activated by diacylglycerol (DAG), have been put forward as possible candidates. Because human embryonic kidney (HEK) 293 cells endogenously express these TRP subunits, this cell line is suitable for investigating whether DAG-activated TRP subunits form part of the putative multimeric assemblies that mediate CCE. Adenylyl cyclase type 8 (AC8) is activated by CCE in nonexcitable cells but is not responsive to other forms of Ca(2+) entry, such as ionophore- or arachidonate-activated entry through the plasma membrane. In this study, we exploited this unique dependence of AC8 on CCE to determine whether the DAG analog, 1-oleyl-2-acetyl-sn-glycerol (OAG), activates the same subset of Ca(2+) channels as store depletion, which triggers CCE. In populations of HEK 293 cells, OAG evoked a faster and greater influx of Ca(2+) than CCE. Both pathways of Ca(2+) entry could be triggered simultaneously in the same batch of cells, with additive effects. It is striking that OAG-mediated Ca(2+) entry, unlike CCE, did not stimulate AC8 activity in populations of cells. In single cells, OAG evoked a highly heterogeneous response, whereas CCE occurred as a smooth and sustained increase in [Ca(2+)](i). Taken together, our results indicate that, in HEK 293 cells, OAG-activated Ca(2+) entry is distinct from CCE. The inability of the OAG-activated Ca(2+) entry pathway to regulate AC8 further reinforces the absolute dependence of this enzyme on CCE.     

The Ca(2+) sensor stromal interaction molecule 1 (STIM1) is necessary and sufficient for the store-operated Ca(2+) entry function of transient receptor potential canonical (TRPC) 1 and 4 channels in endothelial cells

We addressed the requirement for stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+)-sensor, and Orai1, a Ca(2+) selective channel, in regulating Ca(2+) entry through the store-operated channels mouse transient receptor potential canonical (TRPC) 4 or human TRPC1. Studies were made using murine and human lung endothelial cells (ECs) challenged with thrombin known to induce Ca(2+) entry via TRPC1/4. Deletion or knockdown of TRPC4 abolished Ca(2+) entry secondary to depletion of ER Ca(2+) stores, preventing the disruption of the endothelial barrier. Knockdown of STIM1 (but not of Orai1or Orai3) or expression of the dominant-negative STIM1(K684E-K685E) mutant in ECs also suppressed Ca(2+) entry secondary to store depletion. Ectopic expression of WT-STIM1 or WT-Orai1 in TRPC4(-/-)-ECs failed to rescue Ca(2+) entry; however, WT-TRPC4 expression in TRPC4(-/-)-ECs restored Ca(2+) entry indicating the requirement for TRPC4 in mediating store-operated Ca(2+) entry. Moreover, expression of the dominant-negative Orai1(R91W) mutant or Orai3(E81W) mutant in WT-ECs failed to prevent thrombin-induced Ca(2+) entry. In contrast, expression of the dominant-negative TRPC4(EE647-648KK) mutant in WT-ECs markedly reduced thrombin-induced Ca(2+) entry. In ECs expressing YFP-STIM1, ER-store Ca(2+) depletion induced formation of fluorescent membrane puncta in WT but not in TRPC4(-/-) cells, indicating that mobilization of STIM1 and engagement of its Ca(2+) sensing function required TRPC4 expression. Coimmunoprecipitation studies showed coupling of TRPC1 and TRPC4 with STIM1 on depletion of ER Ca(2+) stores. Thus, TRPC1 and TRPC4 can interact with STIM1 to form functional store-operated Ca(2+)-entry channels, which are essential for mediating Ca(2+) entry-dependent disruption of the endothelial barrier.     

Effects of phosphoinositide 3-kinase on endothelin-1-induced activation of voltage-independent Ca2+ channels and vasoconstriction

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2.     

TRPC channels: integrators of multiple cellular signals

TRPC channels are ubiquitously expressed among cell types and mediate signals in response to phospholipase C (PLC)-coupled receptors. TRPC channels function as integrators of multiple signals resulting from receptor-induced PLC activation, which catalyzes the breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 depletes Ca2+ stores and TRPC3 channels can be activated by store-depletion. InsP3 also activates the InsP3 receptor, which may undergo direct interactions with the TRPC3 channel, perhaps mediating store-dependence. The other PLC product, DAG, has a direct non-PKC-dependent activating role on TRPC3 channels likely by direct binding. DAG also has profound effects on the TRPC3 channel through PKC. Thus PKC is a powerful inhibitor of most TRPC channels and DAG is a dual regulator of the TRPC3 channel. PLC-mediated DAG results in rapid channel opening followed later by a slower DAG-induced PKC-mediated deactivation of the channel. The decreased level of PIP2 from PLC activation also has an important modifying action on TRPC3 channels. Thus, the TRPC3 channel and PLCgamma form an intermolecular PH domain that has high specificity for binding PIP2. This interaction allows the channel to be retained within the plasma membrane, a further operational control factor for TRPC3. As nonselective cation channels, TRPC channel opening results in the entry of both Na+ and Ca2+ ions. Thus, while they may mediate Ca2+ entry signals, TRPC channels are also powerful modifiers of membrane potential.     

Source of the elevation Ca2+ evoked by 15-HETE in pulmonary arterial myocytes

We have previously reported that 15-hydroxyeicosatetraenoic acid (15-HETE), a metabolite of arachidonic acid by 15-lipoxygenase, causes pulmonary vasoconstriction via increasing the intracellular Ca(2+) concentration ([Ca(2+)]i). However, the multiple sources of Ca(2+) that contribute to Ca(2+) elevation during and after 15-HETE exposure have not been investigated. In the present study, pulmonary arterial ring technique and confocal laser scanning microscope were used to investigate the origin of Ca(2+). 15-HETE (1 microM) elicited an increase in [Ca(2+)]i in pulmonary artery smooth muscle cells in a time-dependent manner under both normal and hypoxic condition. The increases were composed of an initial rapid rise followed by a slow increase in the present of extracellular Ca(2+). The initial rapid phase was attenuated by inositol 1,4,5-triphosphate (IP(3)) receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) and ryanodine receptor-operated Ca(2+) store depletion agent caffeine; the slow increasing phase and the constriction of pulmonary arterial ring were significantly inhibited by voltage-operated Ca(2+) channel blocker nifedipine or transient receptor potential canonical (TRPC) channel blocker La(3+), and almost completely diminished in Ca(2+)-free external solution, suggesting that the initial phase depends on intracellular Ca(2+) store and the second phase relies on extracellular Ca(2+). Interestingly, the effect of caffeine and La(3+) but not nifedipine were diminished in the present of 2-APB. Thus, these results suggest that 15-HETE mobilizes Ca(2+) signaling through: 1) Ca(2+) release immediately from Ca(2+) stores via activation of IP(3) receptor and, subsequently that of ryanodine receptor, 2) the depletion of Ca(2+) through CCE leading to the activation of TRPC, and 3) Ca(2+) entry through L-type Ca(2+) channels.     

Slow depletion of endoplasmic reticulum Ca(2+) stores and block of store-operated Ca(2+) channels by 2-aminoethoxydiphenyl borate in mouse pancreatic acinar cells

The compound 2-aminoethoxydiphenyl borate (2-APB) has been used as either a specific membrane-permeable inhibitor for InsP(3) receptors or a store-operated Ca(2+) channel blocker in some cells. In this study, we have investigated actions of 2-APB on Ca(2+) signalling in mouse pancreatic acinar cells by measuring Ca(2+) concentration in the cytosol ([Ca(2+)]c) and in the endoplasmic reticulum (ER). Although 2-APB (50 microM) inhibited or modulated [Ca(2+)]c oscillations generated by a low dose of ACh, it did not block InsP(3)-mediated Ca(2+) release from the ER elicited by high doses of acetylcholine (ACh). 2-APB alone more than 70 microM tended to increase [Ca(2+)]c. When we directly measured Ca(2+) concentration in the lumen of the ER with a low affinity Ca(2+) dye, Mag-fluo-4, 2-APB itself slowly lowered ER Ca(2+) concentration and ACh could further release Ca(2+) from the ER in the presence of 2-APB, suggesting its lack of potency to block InsP(3) receptors. When store-operated Ca(2+) entry was evoked by addition of external Ca(2+) (5 mM) after depletion of Ca(2+) stores, 2-APB (50 microM) substantially blocked the Ca(2+) influx in a reversible manner. We conclude that (a) 2-APB is a good blocker for store-operated Ca(2+) channels, (b) 2-APB could not effectively block InsP(3) receptors, and (c) low doses of 2-APB lower Ca(2+) concentration in the lumen of the ER without a significant elevation of [Ca(2+)]c.     

Negative regulation of TRPC3 channels by protein kinase C-mediated phosphorylation of serine 712

TRPC3 is a nonselective cation channel member of the "canonical" transient receptor potential (TRPC) family whose members are activated by phospholipase C-coupled receptors. TRPC3 can be activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) in a protein kinase C-independent manner. On the other hand, phorbol 12-myristate 13-acetate (PMA) inhibits OAG-mediated TRPC3 channel activation, suggesting that phosphorylation of TRPC3 by protein kinase C is a mechanism of receptor-mediated negative feedback. Here, we show PMA-induced phosphorylation of TRPC3 channels in vivo. We demonstrate by site-directed mutagenesis that a single site containing Ser(712) and conserved among all members of the TRPC family is essential for protein kinase C-mediated negative regulation of TRPC3. In human embryonic kidney 293 cells expressing a TRPC3 mutant in which Ser(712) was replaced by alanine (S712A), PMA failed to block channel activation, whereas wild-type TRPC3 activity was completely inhibited. Phosphorylation of the S712A TRPC3 mutant was not stimulated in response to PMA treatment. Furthermore, S712A TRPC3 mutant-mediated Ca(2+) entry after methacholine activation was significantly greater than that of wild-type TRPC3. These findings demonstrate a dual role for phospholipase C-generated diacylglycerol, which serves as a signal for TRPC3 activation as well as a signal for negative feedback via protein kinase C-mediated phosphorylation.     

The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in PC12 cells

We studied the effects of carmustine (1,3-bis(2-chloroethyl)-1-nitrosourea) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells using fura-2 fluorescence imaging. Carmustine (100 microM) caused a delayed increase in [Ca(2+)](i) that developed within approximately 3 h. This effect was enhanced in cells that were pretreated with an inhibitor of glutathione (GSH) synthesis, buthionine sulfoximine (BSO, 200 microM, 24 h), and was suppressed in cells that were treated with an antioxidant deferoxamine (50 microM). The carmustine-induced increase in [Ca(2+)](i) was absolutely dependent on the presence of extracellular Ca(2+) and could be inhibited by dihydropyridine blockers of L-type voltage-gated Ca(2+) channels (nimodipine or nitrendipine, 10 microM). The increase in [Ca(2+)](i) was also suppressed in Cl(-)-free solution and in the presence of the Cl(-) channel blockers, indanyloxyacetic acid 94 (IAA-94, 100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 microM). The inhibition was complete when the blockers were applied simultaneously with carmustine and was partial when the blockers were applied after the initial increase in [Ca(2+)](i). We conclude that carmustine induces an influx of extracellular Ca(2+) through L-type Ca(2+) channels and that this effect is mediated by oxidative stress that results from the depletion of GSH following the inhibition by carmustine of glutathione reductase.     

Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells

The ATP-sensitive K(+) (K(ATP)) channels are composed of sulfonylurea receptor and inwardly rectifying K(+) channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K(ATP)-channel activity in pituitary GH(3) cells. Steady-state fluctuation analyses of macroscopic K(+) current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K(ATP)-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca(2+) measurements revealed the ability of DEPC to decrease Ca(2+) oscillations in GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells.     

Cellular mechanisms of acrolein-induced alteration in calcium signaling in airway smooth muscle

Acrolein, an unsaturated aliphatic aldehyde, is a potent respiratory irritant. We have previously observed that acrolein administered ex vivo to isolated airways alters subsequent airway responsiveness to muscarinic agonists in terms of both mechanical activity of rings and calcium signaling in isolated cells. In the present study, we have examined the mechanisms by which acrolein alters Ca(2+) signaling. In freshly isolated rat tracheal smooth muscle cells, preexposure to acrolein increased the [Ca(2+)](i) oscillation frequency in response to endothelin 1 (ET-1, 0.1 microM), a contractile agonist that acts via the activation of a receptor different from the muscarinic cholinoceptor. We then studied acrolein-induced alteration in cell signaling with special attention to the steps downstream of membrane receptor activation i.e., the inositol 1,4,5-trisphosphate (InsP(3)) signaling pathway. Pretreatment of cells with LiCl (20 mM), a modulator of InsP(3) concentration, mimicked the effect of acrolein exposure on agonist-induced [Ca(2+)](i) response, i.e., increased the amplitude of the first Ca(2+) rise and the oscillation frequency in response to 0.1 and 10 microM acetylcholine (ACh), respectively. Moreover, in tracheal smooth muscle, preexposure to acrolein significantly increased carbachol-induced [(3)H]inositol-phosphates accumulation, up to 34 +/- 11% above unexposed tissue values. Finally, in beta-escin permeabilized cells, injection of InsP(3) (0.1-10 microM) induced a concentration-dependent [Ca(2+)](i) rise followed, for high InsP(3) concentration, by [Ca(2+)](i) oscillations, a calcium response whose pattern was similar to that induced by ACh. Exposure to acrolein did not alter the InsP(3)-induced [Ca(2+)](i) response. These results indicate that the effect of acrolein exposure on Ca(2+) responses in airway smooth muscle is not restricted to activation of the muscarinic cholinoceptor and is due to an enhancement in agonist-induced InsP(3) production. Since acrolein does not modify InsP(3) receptor channel sensitivity, we conclude that acrolein-induced alteration in calcium signaling can be ascribed to its sole effect on InsP(3) production.     

Characterization of noradrenaline-induced increases in intracellular Ca2+ levels in Chinese hamster ovary cells stably expressing human alpha1A-adrenoceptor

The mechanism for noradrenaline (NA)-induced increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and physiological significance of Na(+) influx through receptor-operated channels (ROCs) and store-operated channels (SOCs) were studied in Chinese hamster ovary (CHO) cells stably expressing human alpha(1A)-adrenoceptor (alpha(1A)-AR). [Ca(2+)](i) was measured using the Ca(2+) indicator fura-2. NA (1 microM) elicited transient and subsequent sustained [Ca(2+)](i) increases, which were inhibited by YM-254890 (G(alphaq/11) inhibitor), U-73122 (phospholipase C (PLC) inhibitor), and bisindolylmaleimide I (protein kinase C (PKC) inhibitor), suggesting their dependence on G(alphaq/11)/PLC/PKC. Both phases were suppressed by extracellular Ca(2+) removal, SK&F 96365 (inhibitor of SOC and nonselective cation channel type-2 (NSCC-2)), LOE 908 (inhibitor of NSCC-1 and NSCC-2), and La(3+) (inhibitor of transient receptor potential canonical (TRPC) channel). Reduction of extracellular Na(+) and pretreatment with KB-R7943, a Na(+)/Ca(2+) exchanger (NCX) inhibitor, inhibited both phases of [Ca(2+)](i) increases. These results suggest that 1) stimulation of alpha(1A)-AR with NA elicits the transient and sustained increases in [Ca(2+)](i) mediated through NSCC-2 that belongs to a TRPC family; 2) Na(+) influx through these channels drives NCX in the reverse mode, causing Ca(2+) influx in exchange for Na(+) efflux; and 3) the G(alphaq/11)/PLC/PKC-dependent pathway plays an important role in the increases in [Ca(2+)](i).     

Pulses of external ATP aid to the synchronization of pancreatic beta-cells by generating premature Ca(2+) oscillations

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.     

Induces vasodilatation of rat mesenteric arteryin vitro mainly by inhibiting receptor-mediated Ca2+-influx and Ca2+-release

The purpose of this study was to investigate the effect of atropine on peripheral vasodilation and the mechanisms involved. The isometric tension of rat mesenteric artery rings was recorded in vitro on a myograph. The results showed that atropine, at concentrations greater than 1 microM, relaxed the noradrenalin (NA)-precontracted rat mesenteric artery in a concentration-dependent manner. Atropine-induced vasodilatation was mediated, in part, by an endothelium-dependent mechanism, to which endothelium-derived hyperpolarizing factor may contribute. Atropine was able to shift the NA-induced concentration-response curve to the right, in a non-parallel manner, suggesting the mechanism of atropine was not mediated via the (alpha1-adrenoreceptor. The beta-adrenoreceptor and ATP sensitive potassium channel, a voltage dependent calcium channel, were not involved in the vasodilatation. However, atropine inhibited the contraction derived from NA and CaCI2 in Ca(2+)-free medium, in a concentration dependent manner, indicating the vasodilatation was related to the inhibition of extracellular Ca2+ influx through the receptor-operated calcium channels and intracellular Ca2+ release from the Ca2+ store. Atropine had no effect on the caffeine-induced contraction in the artery segments, indicating the inhibition of intracellular Ca2+ release as a result of atropine most likely occurs via the IP3 pathway rather than the ryanodine receptors. Our results suggest that atropine-induced vasodilatation is mainly from artery smooth muscle cells due to inhibition of the receptor-mediated Ca(2+)-influx and Ca(2+)-release, and partly from the endothelium mediated by EDHF.