Serotonin-induced activation of TRPV4-like current in rat intrapulmonary arterial smooth muscle cells

In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5-hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca(2+) fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 microM) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni(2+) and to ruthenium red, a TRPV channel blocker, and mimicked by 4alpha-phorbol-12,13-didecanoate (4alpha-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4alpha-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension.     

Ca2+ channels involved in endothelin-induced mitogenic response in carotid artery vascular smooth muscle cells

Endothelin (ET)-1 activates twotypes of Ca²⁺-permeable nonselective cation channels(NSCC-1 and NSCC-2) and a store-operated Ca²⁺ channel(SOCC) in rabbit internal carotid artery (ICA) vascular smooth musclecells (VSMCs) in addition to the voltage-operated Ca²⁺channel (VOCC). These channels can be discriminated using the Ca²⁺ channel blockers SK&F-96365 and LOE-908. SK&F-96365 issensitive to NSCC-2 and SOCC, and LOE-908 is sensitive to NSCC-1 andNSCC-2. On the basis of sensitivity to nifedipine, a specific blocker of the L-type VOCC, VOCCs have a minor role in ET-1-inducedmitogenesis. Both LOE-908 and SK&F-96365 inhibited ET-1-inducedmitogenesis in a concentration-dependent manner, and the combination ofLOE-908 and SK&F-96365 abolished it. The IC₅₀ values ofthese blockers for ET-1-induced mitogenesis correlated well with thoseof the ET-1-induced intracellular free Ca²⁺concentration responses. These results indicate that the inhibitory action of these blockers on ET-1-induced mitogenesis may bemediated by blockade of NSCC-1, NSCC-2, and SOCC. Collectively,extracellular Ca²⁺ influx through NSCC-1, NSCC-2, and SOCCmay be essential for ET-1-induced mitogenesis in ICA VSMCs.

     

Invited review: effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo.

To study the effects of flow on in situ endothelial intracellular calcium concentration ([Ca(2+)](i)) signaling, rat aortic rings were loaded with fura 2, mounted on a tissue flow chamber, and divided into control and flow-pretreated groups. The latter was perfused with buffer at a shear stress of 50 dyns/cm(2) for 1 h. Endothelial [Ca(2+)](i) responses to ACh or shear stresses were determined by ratio image analysis. Moreover, ACh-induced [Ca(2+)](i) elevation responses were measured in a calcium-free buffer, or in the presence of SKF-96365, to elucidate the role of calcium influx in the flow effects. Our results showed that 1) ACh increased endothelial [Ca(2+)](i) in a dose-dependent manner, and these responses were incremented by flow-pretreatment; 2) the differences in ACh-induced [Ca(2+)](i) elevation between control and flow-pretreated groups were abolished by SKF-96365 or by Ca(2+)-free buffer; and 3) in the presence of 10(-5) M ATP, shear stress induced dose-dependent [Ca(2+)](i) elevation responses that were not altered by flow-pretreatment. In conclusion, flow-pretreatment augments the ACh-induced endothelial calcium influx in rat aortas ex vivo.     

Cell culture alters Ca2+ entry pathways activated by store-depletion or hypoxia in canine pulmonary arterial smooth muscle cells

Previous studies have shown that, in acutely dispersed canine pulmonary artery smooth muscle cells (PASMCs), depletion of both functionally independent inositol 1,4,5-trisphosphate (IP(3))- and ryanodine-sensitive Ca(2+) stores activates capacitative Ca(2+) entry (CCE). The present study aimed to determine if cell culture modifies intracellular Ca(2+) stores and alters Ca(2+) entry pathways caused by store depletion and hypoxia in canine PASMCs. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured in fura 2-loaded cells. Mn(2+) quench of fura 2 signal was performed to study divalent cation entry, and the effects of hypoxia were examined under oxygen tension of 15-18 mmHg. In acutely isolated PASMCs, depletion of IP(3)-sensitive Ca(2+) stores with cyclopiazonic acid (CPA) did not affect initial caffeine-induced intracellular Ca(2+) transients but abolished 5-HT-induced Ca(2+) transients. In contrast, CPA significantly reduced caffeine- and 5-HT-induced Ca(2+) transients in cultured PASMCs. In cultured PASMCs, store depletion or hypoxia caused a transient followed by a sustained rise in [Ca(2+)](i). The transient rise in [Ca(2+)](i) was partially inhibited by nifedipine, whereas the nifedipine-insensitive transient rise in [Ca(2+)](i) was inhibited by KB-R7943, a selective inhibitor of reverse mode Na(+)/Ca(2+) exchanger (NCX). The nifedipine-insensitive sustained rise in [Ca(2+)](i) was inhibited by SKF-96365, Ni(2+), La(3+), and Gd(3+). In addition, store depletion or hypoxia increased the rate of Mn(2+) quench of fura 2 fluorescence that was also inhibited by these blockers, exhibiting pharmacological properties characteristic of CCE. We conclude that cell culture of canine PASMCs reorganizes IP(3) and ryanodine receptors into a common intracellular Ca(2+) compartment, and depletion of this store or hypoxia activates voltage-operated Ca(2+) entry, reverse mode NCX, and CCE.     

Capacitative Ca(2+) entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells

We investigated the role of capacitative Ca(2+) entry and tyrosine kinase activation in mediating phenylephrine (PE)-induced oscillations in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in canine pulmonary arterial smooth muscle cells (PASMCs). [Ca(2+)](i) was measured as the 340- to 380-nm ratio in individual fura 2-loaded PASMCs. Resting [Ca(2+)](i) was 96 +/- 4 nmol/l. PE (10 micromol/l) stimulated oscillations in [Ca(2+)](i), with a peak amplitude of 437 +/- 22 nmol/l and a frequency of 1.01 +/- 0.12/min. Thapsigargin (1 micromol/l) was used to deplete sarcoplasmic reticulum (SR) Ca(2+) after extracellular Ca(2+) was removed. Under these conditions, a nifedipine-insensitive, sustained increase in [Ca(2+)](i) (140 +/- 7% of control value) was observed when the extracellular Ca(2+) concentration was restored; i.e., capacitative Ca(2+) entry was demonstrated. Capacitative Ca(2+) entry also refilled SR Ca(2+) stores. Capacitative Ca(2+) entry was attenuated (32 +/- 3% of control value) by 50 micromol/l of SKF-96365 (a nonselective Ca(2+)-channel inhibitor). Tyrosine kinase inhibition with tyrphostin 23 (100 micromol/l) and genistein (100 micromol/l) also inhibited capacitative Ca(2+) entry to 63 +/- 12 and 85 +/- 4% of control values, respectively. SKF-96365 (30 micromol/l) attenuated both the amplitude (15 +/- 7% of control value) and frequency (50 +/- 21% of control value) of PE-induced Ca(2+) oscillations. SKF-96365 (50 micromol/l) abolished the oscillations. Tyrphostin 23 (100 micromol/l) also inhibited the amplitude (17 +/- 7% of control value) and frequency (45 +/- 9% of control value) of the oscillations. Genistein (30 micromol/l) had similar effects. Both SKF-96365 and tyrphostin 23 attenuated PE-induced contraction in isolated pulmonary arterial rings. These results demonstrate that capacitative Ca(2+) entry is present and capable of refilling SR Ca(2+) stores in canine PASMCs and may be involved in regulating PE-induced Ca(2+) oscillations. A tyrosine kinase is involved in the signal transduction pathway for alpha(1)-adrenoreceptor activation in PASMCs.     

Characterization of Ca2+ channels involved in ET-1-induced transactivation of EGF receptors

The purpose of this study was to demonstrate the involvement of Ca(2+) influx through voltage-independent Ca(2+) channels (VICCs) in endothelin-1 (ET-1)-induced transactivation of epidermal growth factor receptor protein tyrosine kinase (EGFR PTK) using the Ca(2+) channel blockers LOE-908 and SK&F-96365 in rabbit internal carotid artery vascular smooth muscle cells. ET-1-induced EGFR PTK transactivation was completely inhibited by AG-1478, which is a specific inhibitor of EGFR PTK. In the absence of extracellular Ca(2+), the magnitude of EGFR PTK transactivation was near the basal level. Based on sensitivity to nifedipine, which is a specific blocker of voltage-operated Ca(2+) channels (VOCCs), VOCCs have minor roles in EGFR PTK transactivation. In contrast, Ca(2+) influx through VICCs plays an important role in EGFR PTK transactivation. Moreover, based on the sensitivity of VICCs to SK&F-96365 and LOE-908, VICCs were shown to consist of two types of Ca(2+)-permeable nonselective cation channels (NSCCs), which are designated NSCC-1 and NSCC-2, and a store-operated Ca(2+) channel. In summary, Ca(2+) influx through VICCs plays an essential role in ET-1-induced EGFR PTK transactivation in rabbit internal carotid artery vascular smooth muscle cells.     

Ca2+ sensitization during sustained hypoxic pulmonary vasoconstriction is endothelium dependent

The main aim of this study was to determine the effects of endothelium removal on tension and intracellular Ca(2+) ([Ca(2+)](i)) during hypoxic pulmonary vasoconstriction (HPV) in rat isolated intrapulmonary arteries (IPA). Rat IPA and mesenteric arteries (MA) were mounted on myographs and loaded with the Ca(2+)-sensitive fluorophore fura PE-3. Arteries were precontracted with prostaglandin F(2alpha), and the effects of hypoxia were examined. HPV in isolated IPA consisted of a transient constriction superimposed on a second sustained phase. Only the latter phase was abolished by endothelial denudation. However, removal of the endothelium had no effect on [Ca(2+)](i) at any point during HPV. The endothelin-1 antagonists BQ-123 and BQ-788 did not affect HPV, although constriction induced by 100 nM endothelin-1 was abolished. In MA, hypoxia induced an initial transient rise in tension and [Ca(2+)](i), followed by vasodilatation and a fall in [Ca(2+)](i) to (but not below) prehypoxic levels. These results are consistent with sustained HPV being mediated by an endothelium-derived constrictor factor that is distinct from endothelin-1 and that elicits vasoconstriction via Ca(2+) sensitization.     

Inhibition of hypoxic pulmonary vasoconstriction by antagonists of store-operated Ca2+ and nonselective cation channels

Previous studies indicated that acute hypoxia increased intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) influx, and capacitative Ca(2+) entry (CCE) through store-operated Ca(2+) channels (SOCC) in smooth muscle cells from distal pulmonary arteries (PASMC), which are thought to be a major locus of hypoxic pulmonary vasoconstriction (HPV). Moreover, these effects were blocked by Ca(2+)-free conditions and antagonists of SOCC and nonselective cation channels (NSCC). To test the hypothesis that in vivo HPV requires CCE, we measured the effects of SOCC/NSCC antagonists (SKF-96365, NiCl(2), and LaCl(3)) on pulmonary arterial pressor responses to 2% O(2) and high-KCl concentrations in isolated rat lungs. At concentrations that blocked CCE and [Ca(2+)](i) responses to hypoxia in PASMC, SKF-96365 and NiCl(2) prevented and reversed HPV but did not alter pressor responses to KCl. At 10 microM, LaCl(3) had similar effects, but higher concentrations (30 and 100 microM) caused vasoconstriction during normoxia and potentiated HPV, indicating actions other than SOCC blockade. Ca(2+)-free perfusate and the voltage-operated Ca(2+) channel (VOCC) antagonist nifedipine were potent inhibitors of pressor responses to both hypoxia and KCl. We conclude that HPV required influx of Ca(2+) through both SOCC and VOCC. This dual requirement and virtual abolition of HPV by either SOCC or VOCC antagonists suggests that neither channel provided enough Ca(2+) on its own to trigger PASMC contraction and/or that during hypoxia, SOCC-dependent depolarization caused secondary activation of VOCC.     

Ca(2+) channels activated by endothelin-1 in CHO cells expressing endothelin-A or endothelin-B receptors

We comparedthe Ca²⁺ channels activated by endothelin-1 (ET-1) inChinese hamster ovary (CHO) cells stably expressing endothelin type A(ET_A) or endothelin type B (ET_B) receptorsusing the Ca²⁺ channel blockers LOE-908 and SK&F-96365. Inboth CHO-ET_A and CHO-ET_B, ET-1 at 0.1 nMactivated the Ca²⁺-permeable nonselective cation channel-1(NSCC-1), which was sensitive to LOE-908 and resistant to SK&F-96365.ET-1 at 1 nM activated NSCC-2 in addition to NSCC-1; NSCC-2 wassensitive to both LOE-908 and SK&F-96365. ET-1 at 10 nM activated thesame channels as 1 nM ET-1 in both cell types, but inCHO-ET_A, it additionally activated the store-operatedCa²⁺ channel (SOCC), which was resistant to LOE-908 andsensitive to SK&F-96365. Up to 1 nM ET-1, the level of the formation of inositol phosphates (IPs) was low and similar in both cell types, but,at 10 nM ET-1, it was far greater in CHO-ET_A than inCHO-ET_B. These results show that, in CHO-ET_Aand CHO-ET_B, ET-1 up to 10 nM activated the sameCa²⁺ entry channels: 0.1 nM ET-1 activated NSCC-1, andET-1  1 nM activated NSCC-1 and NSCC-2. Notably, inCHO-ET_A, 10 nM ET-1 activated SOCCs because of the higherformation of IPs.

     

Enhancement of myofilament calcium sensitivity by acute hypoxia in rat distal pulmonary arteries

Hypoxic contraction of pulmonary arterial smooth muscle is thought to require increases in both intracellular Ca(2+) concentration ([Ca(2+)](i)) and myofilament Ca(2+) sensitivity, which may or may not be endothelium-dependent. To examine the effects of hypoxia and endothelium on Ca(2+) sensitivity in pulmonary arterial smooth muscle, we measured the relation between [Ca(2+)](i) and isometric force at 37°C during normoxia (21% O(2)-5% CO(2)) and after 30 min of hypoxia (1% O(2)-5% CO(2)) in endothelium-intact (E+) and -denuded (E-) rat distal intrapulmonary arteries (IPA) permeabilized with staphylococcal α-toxin. Endothelial denudation enhanced Ca(2+) sensitivity during normoxia but did not alter the effects of hypoxia, which shifted the [Ca(2+)](i)-force relation to higher force in E+ and E- IPA. Neither hypoxia nor endothelial denudation altered Ca(2+) sensitivity in mesenteric arteries. In E+ and E- IPA, hypoxic enhancement of Ca(2+) sensitivity was abolished by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (30 μM), which shifted normoxic [Ca(2+)](i)-force relations to higher force. In E- IPA, the Rho kinase antagonist Y-27632 (10 μM) shifted the normoxic [Ca(2+)](i)-force relation to lower force but did not alter the effects of hypoxia. These results suggest that acute hypoxia enhanced myofilament Ca(2+) sensitivity in rat IPA by decreasing nitric oxide production and/or activity in smooth muscle, thereby revealing a high basal level of Ca(2+) sensitivity, due in part to Rho kinase, which otherwise did not contribute to Ca(2+) sensitization by hypoxia.     

KB-R7943 inhibits store-operated Ca(2+) entry in cultured neurons and astrocytes

We have studied cyclopiazonic acid (CPA)-sensitive store-operated Ca(2+) entry (SOCE) in cultured neurons and astrocytes and examined the effect of 2-[2-[4-(4-nitrobenzyloxy)phenyl]]isothiourea (KB-R7943), which is often used as a selective inhibitor of the Na(+)-Ca(2+) exchanger (NCX), on the SOCE. CPA increased transiently intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a sustained increase in [Ca(2+)](i) in neurons and astrocytes. The sustained increase in [Ca(2+)](i) depended on the presence of extracellular Ca(2+) and inhibited by SOCE inhibitors, but not by a Ca(2+) channel inhibitor. CPA also caused quenching of fura-2 fluorescence when the cells were incubated in Mn(2+)-containing medium. KB-R7943 at 10 microM inhibited significantly CPA-induced sustained increase in [Ca(2+)](i) in neurons and astrocytes. KB-R7943 also inhibited CPA-induced quenching of fura-2 fluorescence in the presence of extracellular Mn(2+). These results indicate that cultured neurons and astrocytes possess SOCE and that KB-R7943 inhibits not only NCX but also SOCE.     

Capacitative calcium entry and TRPC channel proteins are expressed in rat distal pulmonary arterial smooth muscle

Mammalian homologs of transient receptor potential (TRP) genes in Drosophila encode TRPC proteins, which make up cation channels that play several putative roles, including Ca2+ entry triggered by depletion of Ca2+ stores in endoplasmic reticulum (ER). This capacitative calcium entry (CCE) is thought to replenish Ca2+ stores and contribute to signaling in many tissues, including smooth muscle cells from main pulmonary artery (PASMCs); however, the roles of CCE and TRPC proteins in PASMCs from distal pulmonary arteries, which are thought to be the major site of pulmonary vasoreactivity, remain uncertain. As an initial test of the possibility that TRPC channels contribute to CCE and Ca2+ signaling in distal PASMCs, we measured [Ca2+]i by fura-2 fluorescence in primary cultures of myocytes isolated from rat intrapulmonary arteries (>4th generation). In cells perfused with Ca2+-free media containing cyclopiazonic acid (10 microM) and nifedipine (5 microM) to deplete ER Ca2+ stores and block voltage-dependent Ca2+ channels, restoration of extracellular Ca2+ (2.5 mM) caused marked increases in [Ca2+]i whereas MnCl2 (200 microM) quenched fura-2 fluorescence, indicating CCE. SKF-96365, LaCl3, and NiCl2, blocked CCE at concentrations that did not alter Ca2+ responses to 60 mM KCl (IC50 6.3, 40.4, and 191 microM, respectively). RT-PCR and Western blotting performed on RNA and protein isolated from distal intrapulmonary arteries and PASMCs revealed mRNA and protein expression for TRPC1, -4, and -6, but not TRPC2, -3, -5, or -7. Our results suggest that CCE through TRPC-encoded Ca2+ channels could contribute to Ca2+ signaling in myocytes from distal intrapulmonary arteries.     

Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca(2+)](i) in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (>or=4th generation) and maintained under hypoxic conditions ( approximately 25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.     

The contraction of smooth muscle cells of intrapulmonary arterioles is determined by the frequency of Ca2+ oscillations induced by 5-HT and KCl

Increased resistance of the small blood vessels within the lungs is associated with pulmonary hypertension and results from a decrease in size induced by the contraction of their smooth muscle cells (SMCs). To study the mechanisms that regulate the contraction of intrapulmonary arteriole SMCs, the contractile and Ca(2+) responses of the arteriole SMCs to 5-hydroxytrypamine (5-HT) and KCl were observed with phase-contrast and scanning confocal microscopy in thin lung slices cut from mouse lungs stiffened with agarose and gelatin. 5-HT induced a concentration-dependent contraction of the arterioles. Increasing concentrations of extracellular KCl induced transient contractions in the SMCs and a reduction in the arteriole luminal size. 5-HT induced oscillations in [Ca(2+)](i) within the SMCs, and the frequency of these Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained arteriole contraction. By contrast, KCl induced Ca(2+) oscillations that occurred with low frequencies and were preceded by small, localized transient Ca(2+) events. The 5-HT-induced Ca(2+) oscillations and contractions occurred in the absence of extracellular Ca(2+) and were resistant to Ni(2+) and nifedipine but were abolished by caffeine. KCl-induced Ca(2+) oscillations and contractions were abolished by the absence of extracellular Ca(2+) and the presence of Ni(2+), nifedipine, and caffeine. Arteriole contraction was induced or abolished by a 5-HT(2)-specific agonist or antagonist, respectively. These results indicate that 5-HT, acting via 5-HT(2) receptors, induces arteriole contraction by initiating Ca(2+) oscillations and that KCl induces contraction via Ca(2+) transients resulting from the overfilling of internal Ca(2+) stores. We hypothesize that the magnitude of the sustained intrapulmonary SMC contraction is determined by the frequency of Ca(2+) oscillations and also by the relaxation rate of the SMC.     

Ion channels modulating mouse dendritic cell functions

Ca(2+)-mediated signal transduction pathways play a central regulatory role in dendritic cell (DC) responses to diverse Ags. However, the mechanisms leading to increased [Ca(2+)](i) upon DC activation remained ill-defined. In the present study, LPS treatment (100 ng/ml) of mouse DCs resulted in a rapid increase in [Ca(2+)](i), which was due to Ca(2+) release from intracellular stores and influx of extracellular Ca(2+) across the cell membrane. In whole-cell voltage-clamp experiments, LPS-induced currents exhibited properties similar to the currents through the Ca(2+) release-activated Ca(2+) channels (CRAC). These currents were highly selective for Ca(2+), exhibited a prominent inward rectification of the current-voltage relationship, and showed an anomalous mole fraction and a fast Ca(2+)-dependent inactivation. In addition, the LPS-induced increase of [Ca(2+)](i) was sensitive to margatoxin and ICAGEN-4, both inhibitors of voltage-gated K(+) (Kv) channels Kv1.3 and Kv1.5, respectively. MHC class II expression, CCL21-dependent migration, and TNF-alpha and IL-6 production decreased, whereas phagocytic capacity increased in LPS-stimulated DCs in the presence of both Kv channel inhibitors as well as the I(CRAC) inhibitor SKF-96365. Taken together, our results demonstrate that Ca(2+) influx in LPS-stimulated DCs occurs via Ca(2+) release-activated Ca(2+) channels, is sensitive to Kv channel activity, and is in turn critically important for DC maturation and functions.     

Cytosolic phospholipase A2 translocates to forming phagosomes during phagocytosis of zymosan in macrophages

Resident tissue macrophages mediate early innate immune responses to microbial infection. Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is activated in macrophages during phagocytosis of non-opsonized yeast (zymosan) triggering arachidonic acid release and eicosanoid production. cPLA(2)alpha translocates from cytosol to membrane in response to intracellular calcium concentration ([Ca(2+)](i)) increases. Enhanced green fluorescent protein (EGFP)-cPLA(2)alpha translocated to forming phagosomes, surrounding the zymosan particle by 5 min and completely overlapping with early endosome (Rab5) and plasma membrane (F4/80) markers but only partially overlapping with resident endoplasmic reticulum proteins (GRP78 and cyclooxygenase 2). EGFP-cPLA(2)alpha also localized to membrane ruffles during phagocytosis. Zymosan induced an initial high amplitude calcium transient that preceded particle uptake followed by a low amplitude sustained calcium increase. Both phases were required for optimal phagocytosis. Extracellular calcium chelation prevented only the sustained phase but allowed a limited number of phagocytic events, which were accompanied by translocation of cPLA(2)alpha to the phagosome although [Ca(2+)](i) remained at resting levels. The results demonstrate that cPLA(2)alpha targets the phagosome membrane, which may serve as a source of arachidonic acid for eicosanoid production.     

Protection against hypoxia-induced blood-brain barrier disruption: changes in intracellular calcium

Tissue damage after stroke is partly due to disruption of the blood-brain barrier (BBB). Little is known about the role of calcium in modulating BBB disruption. We investigated the effect of hypoxic and aglycemic stress on BBB function and intracellular calcium levels. Bovine brain microvessel endothelial cells were treated with A-23187 to increase intracellular calcium without hypoxia or treated with a calcium chelator (BAPTA) or calcium channel blockers (nifedipine or SKF-96365) and 6 h of hypoxia. A-23187 alone did not increase paracellular permeability. Hypoxia increased intracellular calcium, and hypoxia or hypoxia-aglycemia increased paracellular permeability. Treatment with nifedipine and SKF-96365 increased intracellular calcium under normoglycemic conditions, instead of blocking calcium influx, and was protective against hypoxia-induced BBB disruption under normoglycemia. Protection by nifedipine and SKF-96365 was not due to antioxidant properties of these compounds. These data indicate that increased intracellular calcium alone is not enough to disrupt the BBB. However, increased intracellular calcium after drug treatment and hypoxia suggests a potential mechanism for these drugs in BBB protection; nifedipine and SKF-96365 plus hypoxic stress may trigger calcium-mediated signaling cascades, altering BBB integrity. nifedipine; SKF-96365; ischemia; permeability; fura 2     

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.     

Contribution of both Ca2+ entry and Ca2+ sensitization to the alpha1-adrenergic vasoconstriction of rat penile small arteries

Sympathetic adrenergic nerves maintain the flaccid state of the penis through the tonic release of norepinephrine that contracts trabecular and arterial smooth muscle. Simultaneous measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)) and tension and experiments with alpha-toxin-permeabilized arteries were performed in branches of the rat dorsal penile artery to investigate the intracellular Ca(2+) signaling pathways underlying alpha(1)-adrenergic vasoconstriction. Phenylephrine increased both [Ca(2+)](i) and tension, these increases being abolished by extracellular Ca(2+) removal and reduced by about 50% by the L-type Ca(2+) channel blocker nifedipine (0.3 microM). Non-L-type Ca(2+) entry through store-operated channels was studied by inhibiting the sarcoplasmic reticulum Ca(2+)-ATPase with cyclopiazonic acid (CPA). CPA (30 microM) induced variable phasic contractions that were abolished by extracellular Ca(2+) removal and by the store-operated channels antagonist 2-aminoethoxydiphenyl borate (2-APB, 50 microM) and largely inhibited by nifedipine (0.3 microM). CPA induced a sustained increase in [Ca(2+)](i) that was reduced in a Ca(2+)-free medium. Under conditions of L-type channels blockade, Ca(2+) readmission after store depletion with CPA evoked a sustained and marked elevation in [Ca(2+)](i) not coupled to contraction. 2-APB (50 microM) inhibited the rise in [Ca(2+)](i) evoked by CPA and the nifedipine-insensitive increases in both [Ca(2+)](i) and contraction elicited by phenylephrine. In alpha-toxin-permeabilized penile arteries, activation of G proteins with guanosine 5'-O-(3-thiotriphosphate) and of the alpha(1)-adrenoceptor with phenylephrine both enhanced the myofilament sensitivity to Ca(2+). This Ca(2+) sensitization was reduced by selective inhibitors of PKC, tyrosine kinase (TK), and Rho kinase (RhoK) by 43%, 67%, and 82%, respectively. As a whole, the present data suggest the alpha(1)-adrenergic vasoconstriction in penile small arteries involves Ca(2+) entry through both L-type and 2-APB-sensitive receptor-operated channels, as well as Ca(2+) sensitization mechanisms mediated by PKC, TK, and RhoK. A capacitative Ca(2+) entry coupled to noncontractile functions of the smooth muscle cell is also demonstrated.     

Thrombin-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Thrombin is a procoagulant inflammatory agonist that can disrupt the endothelium-lumen barrier in the lung by causing contraction of endothelial cells and promote pulmonary cell proliferation. Both contraction and proliferation require increases in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this study, we compared the effect of thrombin on Ca(2+) signaling in human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells. Thrombin increased the [Ca(2+)](cyt) in both cell types; however, the transient response was significantly higher and recovered quicker in the PASMC, suggesting different mechanisms may contribute to thrombin-mediated increases in [Ca(2+)](cyt) in these cell types. Depletion of intracellular stores with cyclopiazonic acid (CPA) in the absence of extracellular Ca(2+) induced calcium transients representative of those observed in response to thrombin in both cell types. Interestingly, CPA pretreatment significantly attenuated thrombin-induced Ca(2+) release in PASMC; this attenuation was not apparent in PAEC, indicating that a PAEC-specific mechanism was targeted by thrombin. Treatment with a combination of CPA, caffeine, and ryanodine also failed to abolish the thrombin-induced Ca(2+) transient in PAEC. Notably, thrombin-induced receptor-mediated calcium influx was still observed in PASMC after CPA pretreatment in the presence of extracellular Ca(2+). Ca(2+) oscillations were triggered by thrombin in PASMC resulting from a balance of extracellular Ca(2+) influx and Ca(2+) reuptake by the sarcoplasmic reticulum. The data show that thrombin induces increases in intracellular calcium in PASMC and PAEC with a distinct CPA-, caffeine-, and ryanodine-insensitive release existing only in PAEC. Furthermore, a dynamic balance between Ca(2+) influx, intracellular Ca(2+) release, and reuptake underlie the Ca(2+) transients evoked by thrombin in some PASMC. Understanding of such mechanisms will provide an important insight into thrombin-mediated vascular injury during hypertension.