P2Y2-receptor-mediated activation of a contralateral, lanthanide-sensitive calcium entry pathway in the human airway epithelium

1. Receptor-mediated calcium entry (RMCE) was examined in well-differentiated cultures of normal human bronchial epithelial cells (HBECs). Changes in intracellular free Ca(2+) ([Ca(2+)](i)) were quantified using fluorescence ratio imaging of Fura-2-loaded cells during perfusion with Ca(2+) mobilizing agonists. 2. Initial studies revealed an agonist potency of ATP=uridine triphosphate (UTP) >ADP=uridine diphosphate, consistent with purinergic activation of an apical P2Y(2)-receptor mediating the increase in [Ca(2+)](i) in HBECs. 3. Apical UTP (30 microm) induced a sustained period of elevated [Ca(2+)](i) between 300 and 600 s following agonist stimulation that extracellular Ca(2+) free studies indicated was dominated by Ca(2+) influx. 4. RMCE was inhibited by 100 nm La(3+) (83+/-3%) or Gd(3+) (95+/-7%) (P<0.005, n=4-11) and was partially attenuated by Ni(2+) (1 mm) (58.7+/-5.0%, P<0.005, n=9). 5. RMCE was also partially sensitive (< 25% inhibition, P<0.01) to the cation channel blockers SKF96365 (30 microm) and econazole (30 microm), but was insensitive to both verapamil (1 microm) and ruthenium red (10 microm). 6. Using either a sided Ca(2+) readdition protocol or unilateral La(3+), established that the RMCE pathway was located exclusively on the basolateral membrane. 7. The pharmacological sensitivity of the P2Y(2)-receptor activated Ca(2+) entry pathway in the human airway epithelium is inconsistent with the established profile of TRP channel families and is therefore likely to be of an as-yet uncharacterized molecular identity.     

FcepsilonRI cross-linking-induced actin assembly mediates calcium signalling in RBL-2H3 mast cells

1. To determine the role of actin assembly in the Ca(2+) signalling of mast cells activated by cross-linking of FcepsilonRI, we examined the effects of cytochalasin D, an inhibitor of actin polymerization. 2. In the RBL-2H3 cells, F-actin content was increased by sensitization with anti-dinitrophenol (DNP) IgE. In these cells, cytochalasin D induced oscillatory increases in cytosolic Ca(2+) ([Ca(2+)](i)); these increase were inhibited by jasplakinolide, a stabilizer of actin filaments. 3. In the IgE-sensitized RBL-2H3 cells, DNP-human serum albumin (DNP-HSA) augmented actin assembly. DNP-HSA also increased the production of IP(3), [Ca(2+)](i) and degranulation. Cytochalasin D enhanced all of these DNP-HSA-induced effects. 4. In a Ca(2+)-free solution, DNP-HSA induced a transient increase in [Ca(2+)](i), and this increase was accelerated by cytochalasin D. After cessation of the DNP-HSA-induced Ca(2+) release, the re-addition of Ca(2+) induced a sustained increase in [Ca(2+)](i) through capacitative Ca(2+) entry (CCE), and this increase was enhanced by cytochalasin D. 5 The effect of cytochalasin D in enhancing the CCE activity was prevented by xestospongin C. 6. In contrast, neither the Ca(2+) release nor the CCE activation that was induced by thapsigargin was affected by cytochalasin D. 7. These results suggest that actin de-polymerization stimulates the FcepsilonRI-mediated signalling to augment the release of Ca(2+) from the endoplasmic reticulum in RBL-2H3 cells.     

Contribution of extracellular signal-regulated kinase to UTP-induced interleukin-6 biosynthesis in HaCaT keratinocytes

UTP causes interleukin (IL)-6 production via mRNA expression through P2Y(2)/P2Y(4) receptors in human HaCaT keratinocytes. In the present study, we analyzed the mechanism of UTP-induced IL-6 production in these cells. UTP, an agonist of P2Y(2)/P2Y(4) receptors, induced phosphorylation of extracellular signal-regulated kinase (ERK) in a concentration- and time-dependent manner. PD98059, a MEK (mitogen-activated protein kinase kinase) inhibitor, and BAPTA-AM [O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester], an intracellular Ca(2+) chelator, reduced UTP-induced ERK phosphorylation and IL-6 mRNA expression. 2-APB [(2-aminoethoxy)diphenylborane], an inositol 1,4,5-trisphosphate (IP(3))-receptor antagonist, inhibited UTP-induced IL-6 mRNA expression; and the action of A23187, a Ca(2+) ionophore, resembled the action of UTP. In contrast, protein kinase C (PKC) downregulation and pertussis toxin did not affect UTP-induced IL-6 mRNA expression, suggesting that PKC and G(i) are not involved in the UTP-induced IL-6 production. However, AG1478, an epidermal growth factor (EGF)-receptor inhibitor, partially decreased UTP-induced ERK phosphorylation and IL-6 expression. These results suggest that UTP-induced IL-6 production is in part mediated via phosphorylation of ERK through G(q/11)/IP(3)/[Ca(2+)](i) and transactivation of the EGF receptor.     

Ca(2+) signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells

1. Human embryonic kidney (HEK)-293 cells expressing recombinant G alpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca(2+)] ([Ca(2+)](i)) in response to interleukin-8 (IL-8) following pre-stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. 2. Pre-stimulation of cells with adenosine 5'-triphosphate (ATP) revealed a substantial Ca(2+) signalling component mediated by IL-8 (E(max)=83 +/- 8% of maximal ATP response, pEC(50) of IL-8 response=9.7 +/- 0.1). 3. 1 microM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 microM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca(2+)](i) elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca(2+)](i) rapidly returned to basal levels. 4. Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca(2+)](i) elevation but the effects of UTP (E(max) of IL-8 response increased to 50 +/- 1% of the maximal response to ATP, pEC(50) increased to 9.8 +/- 0.1) were greater than those of 2MeSADP (E(max) increased to 36 +/- 2%, pEC(50) increased to 8.7 +/- 0.2). 5. 5. The potentiation of IL-8-mediated Ca(2+) signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca(2+) signalling was apparent in the absence of extracellular Ca(2+), demonstrating the release of Ca(2+) from intracellular stores. 6. Activation of P2Y1 and P2Y2 receptors also revealed Ca(2+) signalling by an endogenously expressed, G alpha(s)-coupled beta-adrenoceptor. 7. In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca(2+) signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.     

The effect of divalent cations on neuronal nitric oxide synthase activity.

Neuronal nitric oxide synthase (NOS I) is a Ca(2+)/calmodulin-binding enzyme that generates nitric oxide (NO*) and L-citrulline from the oxidation of L-arginine, and superoxide (O(2)*(-)) from the one-electron reduction of oxygen (O(2)). Nitric oxide in particular has been implicated in many physiological processes, including vasodilator tone, hypertension, and the development and properties of neuronal function. Unlike Ca(2+), which is tightly regulated in the cell, many other divalent cations are unfettered and can compete for the four Ca(2+) binding sites on calmodulin. The results presented in this article survey the effects of various divalent metal ions on NOS I-mediated catalysis. As in the case of Ca(2+), we demonstrate that Ni(2+), Ba(2+), and Mn(2+) can activate NOS I to metabolize L-arginine to L-citrulline and NO*, and afford O(2)*(-) in the absence of L-arginine. In contrast, Cd(2+) did not activate NOS I to produce either NO* or O(2)*(-), and the combination of Ca(2+) and either Cd(2+), Ni(2+), or Mn(2+) inhibited enzyme activity. These interactions may initiate cellular toxicity by negatively affecting NOS I activity through production of NO*, O(2)*(-) and products derived from these free radicals.     

Effect of toluene diisocyanate on homeostasis of intracellular-free calcium in human neuroblastoma SH-SY5Y cells

The mechanisms of TDI (2,4-toluene diisocyanate)-induced occupational asthma are not fully established. Previous studies have indicated that TDI induces non-specific bronchial hyperreactivity to methacholine and induces contraction of smooth muscle tissue by activating 'capsaicin-sensitive' nerves resulting asthma. Cytosolic-free calcium ion concentrations ([Ca(2+)](c)) are elevated when either capsaicin acts at vanilloid receptors, or methacholine at muscarinic receptors. This study therefore investigated the effects of TDI on Ca(2+) mobilization in human neuroblastoma SH-SY5Y cells. TDI was found to elevate [Ca(2+)](c) by releasing Ca(2+) from the intracellular stores and extracellular Ca(2+) influx. 500 microM TDI induced a net [Ca(2+)](c) increase of 112+/-8 and 78+/-6 nM in the presence and absence of extracellular Ca(2+), respectively. In Ca(2+)-free buffer, TDI induced Ca(2+) release from internal stores to reduce their Ca(2+) content and this reduction was evidenced by a suppression occurring on the [Ca(2+)](c) rise induced by thapsigargin, ionomycin, and methacholine after TDI incubation. In the presence of extracellular Ca(2+), simultaneous exposure to TDI and methacholine led a higher level of [Ca(2+)](c) compared to single methacholine stimulation, that might explain that TDI induces bronchial hyperreactivity to methacholine. We conclude that TDI is capable of interfering the [Ca(2+)](c) homeostasis including releasing Ca(2+) from internal stores and inducing extracellular Ca(2+) influx. The interaction of this novel character and bronchial hyperreactivity need further investigation.     

Characterization of maleimide-activated Ca2+ entry in neutrophils

N-Ethylmaleimide (NEM), a thio-alkylating agent, concentration-dependently stimulated the elevation of [Ca(2+)](i) in rat neutrophils in the presence of external Ca(2+). This effect was not observed in Ca(2+)-free medium and was abrogated by dithiothreitol pretreatment. The application of NEM after cyclopiazonic acid (CPA) stimulated the store-emptying activation of Ca(2+) entry. Unlike CPA-induced cation entry, NEM showed poor uptake of Ba(2+) and Sr(2+) and did not induce Mn(2+) influx. NEM diminished CPA-induced Mn(2+) influx, an effect that was blocked by dithiothreitol. Both Ni(2+) and La(3+) attenuated the elevation of [Ca(2+)](i) in response to NEM; however, greater resistance was observed to Ni(2+) inhibition of NEM-induced Ca(2+) influx than inhibition of store-operated Ca(2+) entry. Both cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A) and 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), Ca(2+) channel blockers, and calyculin A, an inhibitor of protein serine/threonine phosphatases 1/2, diminished the NEM-induced Ca(2+) entry. Treatment of cells with genistein, a general tyrosine kinase inhibitor, or with wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), phosphatidylinositol 3-kinase inhibitors, had no appreciable inhibitory effects on the action of NEM. However, 2-aminoethyldiphenyl borate, an inositol trisphosphate receptor antagonist, enhanced rather than inhibited the [Ca(2+)](i) change in response to NEM. These results indicate that NEM stimulates Ca(2+) entry and regulates Ca(2+) signaling through direct thiol oxidation, bypassing the cellular signal transduction pathway. The NEM-regulated Ca(2+) signal demonstrates characteristics that distinguish it from the store-emptying operation in neutrophils, and therefore represents two distinct modes of Ca(2+) regulation.     

Physiological evidence for a P2Y receptor responsive to diadenosine polyphosphates in human lung via Ca(2+) release studies in bronchial epithelial cells

P2Y(2) receptors that are activated by the extracellular nucleotides ATP or UTP mediate Cl(-) secretion via an increase in [Ca(2+)](i) (intracellular calcium concentration). Therefore, in the lung of patients suffering from cystic fibrosis, inhalation of aerosolized UTP offers a way to circumvent the defect in Cl(-) secretion by the cystic fibrosis transmembrane conductance regulator. A possible alternative for the relatively unstable UTP in inhalation therapy is the more resistant diadenosine tetraphosphate (Ap(4)A). In human and rat lung membranes, Ap(4)A binds to P2 receptor sites coupled to G proteins. Here, we showed that Ap(4)A caused an increase in [Ca(2+)](i) with an EC(50) of 17 microM in human bronchial epithelial cells (HBE1). The [Ca(2+)](i) rise evoked by ATP and UTP was completely, but that induced by Ap(4)A only partially, caused by release of Ca(2+) from internal stores. Moreover, the potency of Ap(4)A to mobilize Ca(2+) was lower than that of ATP and UTP (EC(50) 1.5 and 1.8 microM, respectively), and the maximal increase in [Ca(2+)](i) was considerably smaller than that after ATP or UTP. In accordance with our previous results providing evidence for a common binding site for various diadenosine polyphosphates in lung membranes, all Ap(n)A analogues tested (n = 3 to 6) caused a comparable [Ca(2+)](i) increase. Homologous or heterologous prestimulation largely diminished the increase in [Ca(2+)](i) found after a second pulse of either UTP or Ap(4)A. Although specific binding characteristics and functional responses of Ap(4)A on lung cells are in favor of a distinct receptor for Ap(4)A, the cross-talk between UTP and Ap(4)A in HBE1 cells and the only slight differences in Ca(2+) mobilization by ATP or UTP and Ap(4)A render it impossible at this point to state unequivocally whether there exists a distinct P2Y receptor specific for diadenosine polyphosphates in lung epithelia or whether Ap(4)A activates one of the nucleotide receptors already described.     

Role of Ca2+ signaling in the regulation of endothelial permeability

The vascular endothelial cell forms a semipermeable barrier between blood and interstitium. Inflammatory mediators such as thrombin and histamine induce vascular leakage defined as increased endothelial permeability to plasma proteins and other solutes. Increased endothelial permeability is the hallmark of inflammatory vascular edema. Inflammatory mediators that bind to heptahelical G protein-coupled receptors (GPCR) trigger increased endothelial permeability by increasing the intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) activates key signaling pathways, which mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. The Ca(2+)-dependent protein kinase C (PKC) isoform, PKC-alpha, plays a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions. The increase in [Ca(2+)](i) induced by a variety of agonists is achieved by the generation of inositol 1,4,5-trisphosphate (IP3), activation of IP3 receptors (IP3R), release of stored intracellular Ca(2+), and Ca(2+) entry through plasma membrane channels. Recent findings demonstrate that IP3-sensitive Ca(2+) store depletion activates plasma membrane cation channels (i.e., store-operated cation channels (SOC) or Ca(2+) release activated channels) to cause Ca(2+) influx in endothelial cells. This mode of Ca(2+) influx is also known as capacitative Ca(2+) entry (CCE). Store-operated Ca(2+) influx signals increase in permeability and nitric oxide (NO) production and provokes changes in gene expression in endothelial cells. Recent studies have established that the Drosophila transient receptor potential (TRP) gene family of channels expressed in endothelial cells can function as SOC. Deletion of one of the TRP homologues, TRPC4, in mouse caused impairment in store-operated Ca(2+) current and Ca(2+) store release activated Ca(2+) influx in aortic and lung endothelial cells (LEC). In TRPC4 knockout (TRPC4(-/-)) mice, acetylcholine-induced endothelium-dependent smooth muscle relaxation was drastically reduced. In addition, TRPC4(-/-) mice LEC exhibited lack of actin stress fiber formation and cell retraction in response to thrombin activation of proteinase-activated receptor-1 (PAR-1) in endothelial cells. The increase in lung microvascular permeability in response to thrombin receptor activation was inhibited in TRPC4(-/-) mice. These results indicate that endothelial TRP channels such as TRPC1 and TRPC4 play an important role in signaling the increase in endothelial permeability.     

Effects of trans- and cis-resveratrol on Ca2+ handling in A7r5 vascular myocytes

Although the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K(+)-induced [Ca(2+)](i) increases and on voltage-activated transmembrane Ca(2+) fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca(2+)](i) in absence of extracellular Ca(2+). In high-K(+) Ca(2+)-free solution, reintroduction of Ca(2+) caused a sustained rise in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K(+)-induced response, a biphasic change in [Ca(2+)](i) was observed: a transient reduction of the plateau (<5 min) followed by an increase (>10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca(2+) currents (I(Ca(L))). In conclusion, resveratrol isomers exert a dual effect on [Ca(2+)](i) handling in A7r5 myocytes: 1) a blockade of I(Ca(L)) and 2) an increase in [Ca(2+)](i) by depletion of intracellular Ca(2+) stores (which interferes with the agonist-induced release of intracellular Ca(2+)) and influx of Ca(2+), mainly due to activation of capacitative Ca(2+) entry, although other Ca(2+)-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.     

Bile acids increase intracellular Ca(2+) concentration and nitric oxide production in vascular endothelial cells

The effects of bile acids on intracellular Ca(2+) concentration [Ca(2+)](i) and nitric oxide production were investigated in vascular endothelial cells. Whole-cell patch clamp techniques and fluorescence measurements of [Ca(2+)](i) were applied in vascular endothelial cells obtained from human umbilical and calf aortic endothelial cells. Nitric oxide released was determined by measuring the concentration of NO(2)(-). Deoxycholic acid, chenodeoxycholic acid and the taurine conjugates increased [Ca(2+)](i) concentration-dependently, while cholic acid showed no significant effect. These effects resulted from the first mobilization of Ca(2+) from an inositol 1,4,5-triphosphate (IP(3))-sensitive store, which was released by ATP, then followed by Ca(2+) influx. Both bile acids and ATP induced the activation of Ca(2+)-dependent K(+) current. Oscillations of [Ca(2+)](i) were occasionally monitored with the Ca(2+)-dependent K(+) current in voltage-clamped cells and Ca(2+) measurements of single cells. The intracellular perfusion of heparin completely abolished the ATP effect, but failed to inhibit the bile acid effect. Deoxycholic acid and chenodeoxycholic acid enhanced NO(2)(-) production concentration-dependently, while cholic acid did not enhance it. The bile acids-induced nitric oxide production was suppressed by N(G)-nitro-L-arginine methyl ester, exclusion of extracellular Ca(2+) or N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide hydrochloride (W-7) and calmidazolium, calmodulin inhibitors. These results provide novel evidence showing that bile acids increase [Ca(2+)](i) and subsequently nitric oxide production in vascular endothelial cells. The nitric oxide production induced by bile acids may be involved in the pathogenesis of circulatory abnormalities in liver diseases including cirrhosis.     

Extracellular ATP-dependent activation of plasma membrane Ca(2+) pump in HEK-293 cells

1. It is well known that extracellular ATP (ATP(o)) elevates the intracellular Ca(2+) concentration ([Ca(2+)](i)) by inducing Ca(2+) influx or mobilizing Ca(2+) from internal stores via activation of purinoceptors in the plasma membrane. This study shows that ATP(o) also activates the plasma membrane Ca(2+) pumps (PMCPs) to bring the elevated [Ca(2+)](i) back to the resting level in human embryonic kidney-293 (HEK-293) cells. 2. The duration of ATP(o)-induced intracellular Ca(2+) transients was significantly increased by PMCP blockers, La(3+) or orthovanadate. In contrast, replacement of extracellular Na(+) with NMDG(+), a membrane-impermeable cation, had no significant effect on duration, thus suggesting that Na(+)/Ca(2+) exchangers do not participate in the ATP(o)-induced Ca(2+) transient. 3. A rapid and significant decrease in [Ca(2+)](i), which was not dependent on extracellular Na(+), was induced by ATP(o) in cells pretreated with thapsigargin (TG). This decrease was blocked by orthovanadate, indicating that it was caused by PMCPs rather than sarco/endoplasmic reticulum Ca(2+) pumps (SERCPs). 4. UTP and ATPgammaS also caused a decrease in [Ca(2+)](i) in cells pretreated with TG, although they were less effective than ATP. The effect of UTP implies the involvement of both P2Y(1) and P2Y(2) receptors, while the effect of ATPgammaS implies no significant role of ectophosphorylation and agonist hydrolysis in the agonist-induced [Ca(2+)](i) decreases. 5. These results point to a role of PMCPs in shaping the Ca(2+) signal and in restoring the resting [Ca(2+)](i) level to maintain intracellular Ca(2+) homeostasis after agonist stimulation.     

Store depletion-induced calcium influx in rat cerebellar astrocytes

1. In rat cerebellar astrocytes, intracellular Ca(2+) store depletion by receptor agonists or sarco(endo)plasmic reticulum Ca(2+) ATPase inhibitors induced a transient increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in the absence of extracellular Ca(2+) and a sustained increase in its presence. 2. After 10 min treatment with thapsigargin, the [Ca(2+)](i) was unaffected by removal of thapsigargin, but fell rapidly to the basal level when extracellular Ca(2+) was removed, suggesting the involvement of capacitative Ca(2+) entry (CCE); this effect was not seen until cells had been exposed to thapsigargin for at least 2 min. 3. Using the whole cell voltage clamp technique, a 60-100 pA inward current was activated by store depletion, the reversal potential ranging from -5 to 0 mV. 4. When extracellular Na(+) was isotonically replaced by Tris, the thapsigargin-induced [Ca(2+)](i) increase was enhanced, while the inward current was reduced, indicating that store-operated Ca(2+) channels were permeable to Na(+); however, they were not permeable to Sr(2+) or Ba(2+). 5. Thapsigargin-induced CCE remained the same in the presence of nifedipine, La(3+) or Cd(2+), while it was inhibited in the presence of SK&F96365. 6. In cerebellar astrocytes, inhibition of protein serine/threonine phosphorylation promoted CCE. 7. In conclusion, in rat cerebellar astrocytes, store depletion activated a CCE via channels which were permeable to Ca(2+) and Na(+) and regulated by phosphorylation.     

The cytoskeleton plays a modulatory role in the association between STIM1 and the Ca2+ channel subunits Orai1 and TRPC1

Store-operated Ca(2+) entry (SOCE) is a major pathway for Ca(2+) influx in non-excitable cells. Recent studies favour a conformational coupling mechanism between the endoplasmic reticulum (ER) Ca(2+) sensor STIM1 and Ca(2+) permeable channels in the plasma membrane to explain SOCE. Previous studies have reported a role for the cytoskeleton modulating the activation of SOCE; therefore, here we have investigated whether the interaction between STIM1 and the Ca(2+) permeable channels is modulated by the actin or microtubular network. In HEK-293 cells, treatment with the microtubular disrupter colchicine enhanced both the activation of SOCE and the association between STIM1 and Orai1 or TRPC1 induced by thapsigargin (TG). Conversely, stabilization of the microtubules by paclitaxel attenuated TG-evoked activation of SOCE and the interaction between STIM1 and the Ca(2+) channels Orai1 and TRPC1, altogether suggesting that the microtubules act as a negative regulator of SOCE. Stabilization of the cortical actin filament layer results in inhibition of TG-evoked both association between STIM1, Orai1 and TRPC1 and SOCE. Interestingly, disruption of the actin filament network by cytochalasin D did not significantly modify TG-evoked association between STIM1 and Orai1 or TRPC1 but enhanced TG-stimulated SOCE. Finally, inhibition of calmodulin by calmidazolium enhances TG-evoked SOCE and disruption of the actin cytoskeleton results in inhibition of TG-evoked association of calmodulin with Orai1 and TRPC1. Thus, we demonstrate that the cytoskeleton plays an essential role in the regulation of SOCE through the modulation of the interaction between their main molecular components.     

Pharmacological characterization of a UTP-sensitive P2Y nucleotide receptor in organ cultured coronary arteries

Our lab has previously demonstrated that organ cultured coronary smooth muscle cells express a nucleotide receptor that is dramatically more responsive to UTP than non-organ cultured cells. Thus, the purpose of this study was to pharmacologically characterize this UTP-sensitive nucleotide receptor. Porcine coronary arteries were organ cultured (serum-free media, 37 degrees C) for 4 days, and fura-2 imaging of single cells was used to measure myoplasmic Ca2+ (Cam) in response to several nucleotide agonists. A concentration-response relationship (0.01-100 microM) was generated to the nucleotide receptor agonists, UTP, UDP, ATP, ADP, and 2-MeSATP. The potency order was UTP > UDP = ATP = ADP = 2-MeSATP, thus, this nucleotide receptor is predominantly UTP-sensitive. The Cam response to 10 microM UTP was attenuated approximately 50% by the nucleotide receptor antagonists (10 and 100 microM), suramin, reactive blue 2, and pyridoxalphosphate-6-azophenyl-2',4'-disulphonoic acid (PPADS). Depletion of the sarcoplasmic reticulum Ca2+ store with thapsigargin completely abolished the UTP-induced Cam response. In addition, the peak UTP-induced Cam increase was almost two-fold higher in a 2-mM Ca2+ solution than a 0-mM Ca2+ solution. This suggests that the UTP-induced Cam response is comprised of both Ca2+ influx and the mobilization of intracellular Ca2+ stores. Pertussis toxin reduced the UTP-induced Cam response 50%, thus, the UTP-induced increase in Cam is mediated, in part, via Gi/o. These data suggest this UTP-sensitive receptor belongs to the P2Y nucleotide receptor family; however, it does not possess pharmacological characteristics associated with any known P2Y receptor subtype.     

Pharmacological profile of store-operated channels in cerebral arteriolar smooth muscle cells

1. In this study, we determined a pharmacological profile of store-operated channels (SOCs) in smooth muscle cells of rabbit pial arterioles. Ca(2+)-indicator dyes, fura-PE3 or fluo-4, were used to track [Ca(2+)](i) and 10 micro M methoxyverapamil (D600) was present in all experiments on SOCs to prevent voltage-dependent Ca(2+) entry. Store depletion was induced using thapsigargin or cyclopiazonic acid. 2. SOC-mediated Ca(2+) entry was inhibited concentration dependently by Gd(3+) (IC(50) 101 nM). It was also inhibited by 10 micro M La(3+) (70% inhibition, N=5), 100 micro M Ni(2+) (57% inhibition, N=5), 75 micro M 2-aminoethoxydiphenylborate (66% inhibition, N=4), 100 micro M capsaicin (12% inhibition, N=3) or preincubation with 10 micro M wortmannin (76% inhibition, N=4). It was completely resistant to 1 micro M nifedipine (N=5), 10 micro M SKF96365 (N=6), 10 micro M LOE908 (N=14), 10-100 micro M ruthenium red (N=1+2), 100 micro M sulindac (N=4), 0.5 mM streptomycin (N=3) or 1 : 10,000 dilution Grammostolla spatulata venom (N=4). 3. RT-PCR experiments on isolated arteriolar fragments showed expression of mRNA species for TRPC1, 3, 4, 5 and 6. 4. The pharmacological profile of SOC-mediated Ca(2+) entry in arterioles supports the hypothesis that these SOCs are distinct from tonically active background channels and several store-operated and other nonselective cation channels described in other cells. Similarities with the pharmacology of TRPC1 support the hypothesis that TRPC1 is a subunit of the arteriolar smooth muscle SOC.     

Estimation of increased concentration of intracellular Cd(2+) by fluo-3 in rat thymocytes exposed to CdCl(2)

Cadmium, an environmental pollutant, has been reported to induce apoptosis in murine lymphocytes. To reveal the mechanism of cadmium-induced apoptosis, one of important questions is whether cadmium increases intracellular concentration of Ca(2+) ([Ca(2+)](i)), Cd(2+) ([Cd(2+)](i)) or both. It is difficult to detect the increase in [Ca(2+)](i) using Ca(2+)-chelator-based fluorescent Ca(2+) indicators in the presence of Cd(2+) because of their sensitivity to Cd(2+). Therefore, the study on membrane response such as Ca(2+)-dependent hyperpolarization gives a clue to reveal whether the [Ca(2+)](i) or [Cd(2+)](i) is increased. Cadmium at concentrations of 3 μM or more dose-dependently augmented fluo-3 fluorescence in rat thymocytes, presumably suggesting an increased [Ca(2+)](i). However, the membranes were not hyperpolarized although the cells possess Ca(2+)-dependent K(+) channels. One may argue that cadmium inhibits Ca(2+)-dependent K(+) channels so that cadmium fails to hyperpolarize the membranes. It is unlikely because the [Ca(2+)](i) increased by A23187, a calcium ionophore, elicited the hyperpolarization in the presence of Cd(2+). Furthermore, the profile of cytotoxicity induced by cadmium, examined by ethidium bromide and annexin V-FITC, was different from that induced by A23187. Taken together, it is concluded that the application of cadmium increases the [Cd(2+)](i) rather than the [Ca(2+)](i) in rat thymocytes, resulting in the induction of cytotoxicity.     

Regulation of the calcium channel alpha(1G) subunit by divalent cations and organic blockers

The pharmacological properties of the expressed murine T-type alpha(1G) channel were characterized using the whole cell patch clamp configuration. Ba(2+) or Ca(2+) were used as charge carriers. Both I(Ba) and I(Ca) were blocked by Ni(2+) and Cd(2+) with IC(50) values of 0.47+/-0.04 and 1.13+/-0.06 mM (Ni(2+)) and 162+/-13 and 658+/-23 microM (Cd(2+)), respectively. Ni(2+), but not Cd(2+), modified the gating of channel activation. Ni(2+) consistently accelerated channel deactivation while Cd(2+) had a similar effect only on I(Ca). The alpha(1G) channel was potently blocked by mibefradil in a dose- and voltage-dependent manner. I(Ba) was moderately blocked by phenytoin (IC(50) 73.9+/-1.9 microM) and was resistant to the block by valproate. Also 3 mM ethosuximide blocked 20 and 35% of the I(Ba) at a HP of -100 and -60 mV, respectively, while 5 mM amiloride inhibited I(Ba) by 38% and significantly slowed current activation. The alpha(1G) channel was not affected by 10 microM tetrodotoxin. Both 1 microM (+)isradipine and 10 microM nifedipine inhibited 18 and 14% of I(Ba) amplitude at a HP of -100 mV, and 23% and 29% of I(Ba) amplitude at a HP of -60 mV, respectively. The alpha(1G) current was minimally activated by 1 microM Bay K 8644.     

Defect in regulation of Ca2+ movement in platelets from patients with systemic lupus erythematosus

The differences in the intracellular Ca(2+) responses to hormones in platelets from systemic lupus erythematosus (SLE) patients compared to normal humans have not been explored. This study examined the Ca(2+) signaling and density of platelets in normal, inactive and active SLE patients. The platelet number per mul in inactive and normal groups did not differ, whereas the number in active SLE patients was smaller than the other two groups by 60%. The intracellular free Ca(2+) levels ([Ca(2+)](i)) in response to stimulation of four endogenous Ca(2+) mobilizing hormones, 100 microM arachidonic acid (AA), 10 microM ADP, 10 nM platelet activation factor (PAF) and 1 microM thrombin, were investigated using the Ca(2+)-sensitive fluorescent dye, fura-2. The AA-induced [Ca(2+)](i) rises in normal and inactive groups were similar. In contrast, the AA-induced [Ca(2+)](i) rises in the active SLE group were significantly smaller than in the normal and inactive groups. The defect in the AA-induced [Ca(2+)](i) rises in active SLE groups appears to be caused by defective Ca(2+) influx and Ca(2+) releasing pathways because the AA-induced responses were not altered by removal of extracellular Ca(2+), whereas the AA-induced responses in normal and inactive SLE groups were reduced by removal of extracellular Ca(2+), and the AA-induced Ca(2+) release was smaller in the active SLE group. PAF, ADP and thrombin all induced [Ca(2+)](i) rises in the three groups, but no significant differences were found among the three groups. Together, the results indicate that cell density and Ca(2+) signaling in platelets from active SLE patients are altered in response to particular stimulators. In these regards, platelets from inactive SLE patients appear to be similar to those from normal humans.     

Mechanism of damnacanthal-induced [Ca(2+)](i) elevation in human dermal fibroblasts

Damnacanthal is a potent and selective inhibitor of p56(lck) tyrosine kinase in a variety of tissues. We have found, however, using the Ca(2+) microfluorimetry technique, that damnacanthal releases intracellular Ca(2+) stores and promotes Ca(2+) entry in human dermal fibroblasts. The effect of damnacanthal on the peak [Ca(2+)](i) values and the latent time to the peak was concentration-dependent. Damnacanthal releases Ca(2+) from thapsigargin-sensitive Ca(2+) stores, and the Ca(2+) stores responding to damnacanthal were overlapped with those of bradykinin. Damnacanthal-induced Ca(2+) entry was mediated by voltage-dependent and voltage-independent Ca(2+) channels. This effect of damnacanthal on intracellular Ca(2+) mobilization was also observed in cultured bovine coronary endothelial cells but not demonstrated in freshly isolated rat basilar smooth muscle cells. Our study suggests that damnacanthal increases intracellular Ca(2+) by releasing Ca(2+) from internal stores and promoting Ca(2+) entry. The relationship between the actions of damnacanthal on tyrosine kinase and intracellular Ca(2+) requires further investigation.