Role of phosphoinositide 3-kinase in the nonselective cation channel activation by endothelin-1/endothelinB receptor

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) in Chinese hamster ovarian cells expressing endothelin(B) receptor (CHO-ET(B)R). These channels can be discriminated using the Ca(2+) channel blockers, LOE 908 and SK&F 96365. LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of NSCC-2. In this study, we investigated the possible role of phosphoinositide 3-kinase (PI3K) in the ET-1-induced activation of NSCCs in CHO-ET(B)R using wortmannin and LY-294002, inhibitors of PI3K. ET-1-induced Ca(2+) influx was partially inhibited in CHO-ET(B)R pretreated with wortmannin or LY-294002. In contrast, addition of wortmannin or LY-294002 after stimulation with ET-1 did not suppress Ca(2+) influx. The Ca(2+) channels activated by ET-1 in wortmannin- or LY-294002-treated CHO-ET(B)R were sensitive to LOE 908 and resistant to SK&F 96365. In conclusion, NSCC-2 is stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated independently of the PI3K pathway. Moreover, PI3K seems to be required for the initiation of the Ca(2+) entry through NSCC-2 but not for its maintenance.     

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.

     

Characterization of G proteins involved in activation of nonselective cation channels and arachidonic acid release by norepinephrine/alpha1A-adrenergic receptors

We demonstrated recently that norepinephrine activates Ca²⁺-permeable nonselective cation channels (NSCCs) in Chinese hamster ovary cells stably expressing _1A-adrenergic receptors (CHO-_1A). Moreover, extracellular Ca²⁺ through NSCCs plays essential roles in norepinephrine-induced arachidonic acid release. The purpose of the present study was to identify the G proteins involved in the activation of NSCCs and arachidonic acid release by norepinephrine. For these purposes, we used U73122, an inhibitor of phospholipase C (PLC), and dominant negative mutants of G₁₂ and G₁₃ (G₁₂G228A and G₁₃G225A, respectively). U73122 failed to inhibit NSCCs activation by norepinephrine. The magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₃G225A were smaller than those in CHO-_1A. In contrast, the magnitudes of norepinephrine-induced extracellular Ca²⁺ influx in CHO-_1A microinjected with G₁₂G228A were similar to those in CHO-_1A. In addition, neither a Rho-associated kinase (ROCK) inhibitor nor a phosphoinositide 3-kinase inhibitor affected norepinephrine-induced extracellular Ca²⁺ influx. G₁₃G225A, but not G₁₂G228A, also inhibited arachidonic acid release partially. These results demonstrate that 1) the G_q/PLC-pathway is not involved in NSCCs activation by norepinephrine, 2) G₁₃ couples with CHO-_1A and plays important roles for norepinephrine-induced NSCCs activation, 3) neither ROCK- nor PI3K-dependent cascade is involved in NSCCs activation, and 4) G₁₃ is involved in norepinephrine-induced arachidonic acid release in CHO-_1A. norepinephrine; _1A-adrenergic receptor; nonselective cation channel; G₁₃ protein; arachidonic acid release     

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.     

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.

     

CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle

In vascular smooth muscle (VSM) and manyother cells, G protein receptor-coupled activation of mitogen-activatedprotein kinases has been linked, in part, to increases in freeintracellular Ca²⁺. Previously, we demonstrated thationomycin-, angiotensin II-, and thrombin-induced activation ofextracellular signal-regulated kinase (ERK)1/2 in VSM cells wasattenuated by pretreatment with KN-93, a selective inhibitor of themultifunctional Ca²⁺/calmodulin-dependent protein kinase(CaM kinase II). In the present study, we show that theCa²⁺-dependent pathway leading to activation of ERK1/2 ispreceded by nonreceptor proline-rich tyrosine kinase (PYK2) activation and epidermal growth factor (EGF) receptor tyrosine phosphorylation andis attenuated by inhibitors of src family kinases or the EGF receptor tyrosine kinase. Furthermore, we demonstrate that pretreatment with KN-93 or a CaM kinase II inhibitor peptide inhibitsCa²⁺-dependent PYK2 activation and EGF receptor tyrosinephosphorylation in response to ionomycin, ATP, and platelet-derivedgrowth factor but has no effect on phorbol 12,13-dibutyrate- orEGF-induced responses. The results implicate CaM kinase II as anintermediate in the Ca²⁺/calmodulin-dependent activation of PYK2.

     

A role for PYK2 in ANG II-dependent regulation of the PHAS-1-eIF4E complex by multiple signaling cascades in vascular smooth muscle

Regulation of the PHAS-1-eukaryotic initiation factor-4E (eIF4E) complex is the rate-limiting step in the initiation of protein synthesis. This study characterized the upstream signaling pathways that mediate ANG II-dependent phosphorylation of PHAS-1 and eIF4E in vascular smooth muscle. ANG II-dependent PHAS-1 phosphorylation was maximal at 10 min (2.47 ± 0.3 fold vs. control). This effect was completely blocked by the specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase, LY-294002), mammalian target of rapamycin, and extracellular signal-regulated kinase 1/2 (ERK1/2, U-0126) or by a recombinant adenovirus encoding dominant-negative Akt. PHAS-1 phosphorylation was followed by dissociation of eIF4E. Increased ANG II-induced eIF4E phosphorylation was observed at 45 min (2.63 ± 0.5 fold vs. control), was maximal at 90 min (3.38 ± 0.3 fold vs. control), and was sustained at 2 h. This effect was blocked by inhibitors of the ERK1/2 and p38 mitogen-activated protein (MAP) kinase pathways, but not by PI3-kinase inhibition, and was dependent on PKC, intracellular Ca²⁺, and tyrosine kinases. Downregulation of proline-rich tyrosine kinase 2 (PYK2) by antisense oligonucleotides led to a near-complete inhibition of PHAS-1 and eIF4E phosphorylation in response to ANG II. Therefore, PYK2 represents a proximal signaling intermediate that regulates ANG II-induced vascular smooth muscle cell protein synthesis via regulation of the PHAS-1-eIF4E complex. tyrosine kinase; antisense oligonucleotides; protein synthesis     

Extensor protoplasts of the Phaseolus pulvinus: light-induced swelling may require extracellular Ca2+ influx, dark-induced shrinking inositol 1,4,5-trisphosphate-induced Ca2+ mobilization

The photonastic upward movement and scotonastic downward movementof the primary leaf of Phaseolus coccineus L. depends on ionfluxes across the plasma membrane of extensor and flexor cellsof the laminar pulvinus. Extensor protoplasts cultured in 0.4M mannitol, 10 mM KCl, 1 mM CaCl₂ and 5 mM MES-KOH buffer pH6 were found to swell upon switching on white light at the endof a 15 h dark period and to shrink upon switching off the lightat the end of the following 9 h light period, behaviour consistentwith that expected in the cells of intact plants. Light-inducedswelling requires Ca²⁺ in the surrounding medium. Both the Ca²⁺channel blocker verapamil and La³⁺ inhibited this reaction,whereas TMB-8, an inhibitor of intracellular Ca²⁺ transport,had no effect. When the Ca²⁺ iono phore A 23187, the Ca²⁺ channelagonist Bay K-8644, or thapsigargin, an inhibitor of Ca²⁺ -ATPasesat endo-membranes, was added to the medium, extensor proto-plastsswelled in the dark. These results suggest that in extensorprotoplasts light opens Ca²⁺ channels in the plasma membraneand that the influx of extracellular Ca²⁺ results in an increasedcytoplasmic Ca²⁺ concentration which is sufficient to mimicthe light-on signal in activating or deactivating the ion transportersrequired for swelling. Dark-induced shrinking occurred in Ca²⁺-free medium. It was not inhibited by verapamil, but was byTMB-8. Both neomycin and Li⁺ , substances which are known toinhibit the phosphoinositide path way of transmembrane signalling,inhibited dark induced shrinking. Myo-inositol nullified theLi⁺ inhibition of dark-induced shrinking. Neither A 23187 norBay K-8644 induced shrinking in the light, but were able tonullify the inhibitory effect of TMB-8 on dark-induced shrinking.These results suggest that, in extensor protoplasts, the shrinkingsignal ‘light off’ is transduced through phosphoinositidehydrolysis and Ca²⁺ release from internal stores. In additionto the inositol 1,4,5-trisphosphate (IP₃)-induced increase ofthe cytoplasmic Ca²⁺ concentration, further events dependingon the light-off signal appear to be required for shrinking. Key words: Phaseolus pulvinus, extensor protoplasts, light-induced swelling, dark-induced shrinking, Ca²⁺, phosphoinositide signalling     

Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells

High concentrations of cytosolic Na⁺ ions induce the time-dependent formation of an inactive state of the Na⁺/Ca²⁺ exchanger (NCX), a process known as Na⁺-dependent inactivation. NCX activity was measured as Ca²⁺ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na⁺-dependent inactivation. As expected, 1) Na⁺-dependent inactivation was promoted by high cytosolic Na⁺ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na⁺-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na⁺-dependent inactivation in the WT exchanger, 3) Na⁺-dependent inactivation did not increase the half-maximal cytosolic Ca²⁺ concentration for allosteric Ca²⁺ activation, 4) Na⁺-dependent inactivation was not reversed by high cytosolic Ca²⁺ concentrations, and 5) Na⁺-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca²⁺ concentration. Thus Na⁺-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na⁺-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca²⁺ influx during ischemia. ischemia; cytosolic calcium concentration; cytosolic sodium concentration; cellular phosphatidylinositol-4,5-bisphosphate     

Effect of Divalent Cations on Na+ Permeability of Chara corallina and Freshwater Grown Chara buckellii

The effect of elevated Na⁺ concentration on Na⁺ permeability(P_Na) and Na⁺ influx in the presence of two levels of externaldivalent cations was determined in Chara corallina and freshwater-culturedChara buckellii. When Na⁺ in the medium was increased from 1.0to 70 mol m^–3, Na⁺ influx increased in both species ifCa²⁺ was low (0.1 mol m–3). If Ca²⁺ was increased to 7.0mol m^–3 when Na⁺ was increased, Na⁺ influx remained atthe low control level in C. corallina, and showed only a temporaryincrease in C. buckellii. Mg²⁺ was a better substitute for Ca²⁺in C. buckellii than in C. corallina. Na⁺ permeability data suggest that when the external Ca²⁺ concentrationis low, P_Na does not increase in the presence of elevated NaCl;the increase in Na⁺ influx appears to be due to the increasein external Na⁺ concentration alone. Ca^{2 +} supplementation appearsto decrease P_Na whereas supplemental Mg²⁺ has no effect. Na⁺ effluxes were computed from previously determined net fluxesand the influxes. It was found that for both species, fluxesin both directions were stimulated in response to all experimentaltreatments, but Na⁺ influx always exceeded efflux. This resultedin net Na⁺ accumulation in the vacuoles of both species. The results are discussed with reference to net flux and electrophysiologicaldata obtained previously under identical conditions, as wellas the comparative salinity tolerance of both species and theNa⁺/divalent cation ratio. Key words: Na⁺ influx, Na⁺ tolerance, membrane potential, permeability, Chara     

Endothelin-1 induces lipolysis through activation of the GC/cGMP/Ca2+/ERK/CaMKIII pathway in 3T3-L1 adipocytes

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide produced and secreted mainly by endothelial cells. Recent studies indicate that ET-1 can regulate lipid metabolism, which may increase the risk of insulin resistance. Our previous studies revealed that ET-1 induced lipolysis in adipocytes, but the underlying mechanisms were unclear. 3T3-L1 adipocytes were used to investigate the effect of ET-1 on lipolysis and the underlying mechanisms. Glycerol levels in the incubation medium and hormone-sensitive lipase (HSL) phosphorylation were used as indices for lipolysis. ET-1 significantly increased HSL phosphorylation and lipolysis, which were completely inhibited by ERK inhibitor (PD98059) and guanylyl cyclase (GC) inhibitor (LY83583). LY83583 reduced ET-1-induced ERK phosphorylation. A Ca²⁺-free medium and PLC inhibitor caused significant decreases in ET-1-induced lipolysis as well as ERK and HSL phosphorylation, and IP₃ receptor activator (D-IP₃) increased lipolysis. ET-1 increased cGMP production, which was not affected by depletion of extracellular Ca²⁺. On the other hand, LY83583 diminished the ET-1-induced Ca²⁺ influx. Transient receptor potential vanilloid-1 (TRPV-1) antagonist and shRNA partially inhibited ET-1-induced lipolysis. ET-1-induced lipolysis was completely suppressed by CaMKIII inhibitor (NH-125). These results indicate that ET-1 stimulates extracellular Ca²⁺ entry and activates the intracellular PLC/IP₃/Ca²⁺ pathway through a cGMP-dependent pathway. The increased cytosolic Ca²⁺ that results from ET-1 treatment stimulates ERK and HSL phosphorylation, which subsequently induces lipolysis. ET-1 induces HSL phosphorylation and lipolysis via the GC/cGMP/Ca²⁺/ERK/CaMKIII signaling pathway in 3T3-L1 adipocytes.     

Involvement of extracellular Ca2+ influx through voltage-independent Ca2+ channels in endothelin-1 function

This article reviews the types and roles of voltage-independent Ca(2+) channels involved in the endothelin-1 (ET-1)-induced functional responses such as vascular contraction, cell proliferation, and intracellular Ca(2+)-dependent signaling pathways and discusses the molecular mechanisms for the activation of voltage-independent Ca(2+) channels by ET-1. ET-1 activates some types of voltage-independent Ca(2+) channels, such as Ca(2+)-permeable nonselective cation channels (NSCCs) and store-operated Ca(2+) channels (SOCC). Extracellular Ca(2+) influx through these voltage-independent Ca(2+) channels plays essential roles in ET-1-induced vascular contraction, cell proliferation, activation of epidermal growth factor receptor tyrosine kinase, regulation of proline-rich tyrosine kinase, and release of arachidonic acid. The experiments using various constructs of endothelin receptors reveal the importance of G(q) and G(12) families in activation of these Ca(2+) channels by ET-1. These findings provide a potential therapeutic mechanism of a functional interrelationship between G(q)/G(12) proteins and voltage-independent Ca(2+) channels in the pathophysiology of ET-1, such as in chronic heart failure, hypertension, and cerebral vasospasm.     

Endothelin-1 decreases glutamate uptake in primary cultured rat astrocytes

Endothelin-1 (ET-1) is a potent vasoconstrictorpeptide that is also known to induce a wide spectrum of biologicalresponses in nonvascular tissue. In this study, we found that ET-1 (100 nM) inhibited the glutamate uptake in cultured astrocytes expressing the glutamate/aspartate transporter (GLAST); astrocytes did not expressthe glutamate transporter-1 (GLT-1). The V_maxand the K_m of the glutamate uptake were reducedby 57% and 47%, respectively. Application of the ET_A andET_B receptor antagonists BQ-123 and BQ-788 partly inhibitedthe ET-1-evoked decrease in the glutamate uptake, whereas thenonspecific ET receptor antagonist bosentan completely inhibited thisdecrease. Incubation of the cultures with pertussis toxin abolished theeffect of ET-1 on the uptake. The ET-1-induced decrease in theglutamate uptake was independent of extracellular free Ca²⁺concentration, whereas the intracellular Ca²⁺ antagoniststhapsigargin and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester abolished the effect of ET-1 on the glutamate uptake. Incubation with the protein kinase C (PKC) antagonist staurosporine, but not withthe fatty acid-binding protein bovine serum albumin, prevented theET-1-induced decrease in the glutamate uptake. These results suggestthat ET-1 impairs the high-affinity glutamate uptake in culturedastrocytes through a G protein-coupled mechanism, involving PKC andchanges in intracellular Ca²⁺.

     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.

     

Na(+) entry via store-operated channels modulates Ca(2+) signaling in arterial myocytes

In manynonexcitable cells, hormones and neurotransmitters activateNa⁺ influx and mobilizeCa²⁺ from intracellular stores.The stores are replenished by Ca²⁺influx via "store-operated"Ca²⁺ channels (SOC). The mainroutes of Na⁺ entry in these cellsare unresolved, and no role forNa⁺ in signaling has beenrecognized. We demonstrate that the SOC are a majorNa⁺ entry route in arterialmyocytes. Unloading of the Ca²⁺stores with cyclopiazonic acid (a sarcoplasmic reticulumCa²⁺ pump inhibitor) and caffeineinduces a large externalNa⁺-dependent rise in thecytosolic Na⁺ concentration. Onecomponent of this rise in cytosolicNa⁺ concentration is likely due toNa⁺/Ca²⁺exchange; it depends on elevation of cytosolicCa²⁺ and is insensitive to 10 mMMg²⁺ and 10 µMLa³⁺. Another component isinhibited by Mg²⁺ andLa³⁺, blockers of SOC; thiscomponent persists in cells preloaded with1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid to buffer Ca²⁺ transients andpreventNa⁺/Ca²⁺exchange-mediated Na⁺ entry. ThisNa⁺ entry apparently is mediatedby SOC. The Na⁺ entry influencesNa⁺ pump activity andNa⁺/Ca²⁺exchange and has unexpectedly large effects on cell-wideCa²⁺ signaling. The SOC pathwaymay be a general mechanism by which Na⁺ participates in signaling inmany types of cells.

     

Different kinases regulate activation of voltage-dependent calcium channels by depolarization in GH3 cells

The L-type Ca²⁺ channel is the primary voltage-dependent Ca²⁺-influx pathway in many excitable and secretory cells, and direct phosphorylation by different kinases is one of the mechanisms involved in the regulation of its activity. The aim of this study was to evaluate the participation of Ser/Thr kinases and tyrosine kinases (TKs) in depolarization-induced Ca²⁺ influx in the endocrine somatomammotrope cell line GH3. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using a spectrofluorometric method with fura 2-AM, and 12.5 mM KCl (K⁺) was used as a depolarization stimulus. K⁺ induced an abrupt spike (peak) in [Ca²⁺]_i that was abolished in the presence of nifedipine, showing that K⁺ enhances [Ca²⁺]_i, preferably activating L-type Ca²⁺ channels. H89, a selective PKA inhibitor, significantly reduced depolarization-induced Ca²⁺ mobilization in a concentration-related manner when it was applied before or after K⁺, and okadaic acid, an inhibitor of Ser/Thr phosphatases, which has been shown to regulate PKA-stimulated L-type Ca²⁺ channels, increased K⁺-induced Ca²⁺ entry. When PKC was activated by PMA, the K⁺-evoked peak in [Ca²⁺]_i, as well as the plateau phase, was significantly reduced, and chelerythrine (a PKC inhibitor) potentiated the K⁺-induced increase in [Ca²⁺]_i, indicating an inhibitory role of PKC in voltage-dependent Ca²⁺ channel (VDCC) activity. Genistein, a TK inhibitor, reduced the K⁺-evoked increase in [Ca²⁺]_i, but, unexpectedly, the tyrosine phosphatase inhibitor orthovanadate reduced not only basal Ca²⁺ levels but, also, Ca²⁺ influx during the plateau phase. Both results suggest that different TKs may act differentially on VDCC activation. Activation of receptor TKs with epidermal growth factor (EGF) or vascular endothelial growth factor potentiated K⁺-induced Ca²⁺ influx, and AG-1478 (an EGF receptor inhibitor) decreased it. However, inhibition of the non-receptor TK pp60 c-Src enhanced K⁺-induced Ca²⁺ influx. The present study strongly demonstrates that a complex equilibrium among different kinases and phosphatases regulates VDCC activity in the pituitary cell line GH3: PKA and receptor TKs, such as vascular endothelial growth factor receptor and EGF receptor, enhance depolarization-induced Ca²⁺ influx, whereas PKC and c-Src have an inhibitory effect. These kinases modulate membrane depolarization and may therefore participate in the regulation of a plethora of intracellular processes, such as hormone secretion, gene expression, protein synthesis, and cell proliferation, in pituitary cells. phosphatases; protein kinase A; protein kinase C; epidermal growth factor     

Depolarization induces Rho-Rho kinase-mediated myosin light chain phosphorylation in kidney tubular cells

Myosin-based contractility plays important roles in the regulation of epithelial functions, particularly paracellular permeability. However, the triggering factors and the signaling pathways that control epithelial myosin light chain (MLC) phosphorylation have not been elucidated. Herein we show that plasma membrane depolarization provoked by distinct means, including high extracellular K⁺, the lipophilic cation tetraphenylphosphonium, or the ionophore nystatin, induced strong diphosphorylation of MLC in kidney epithelial cells. In sharp contrast to smooth muscle, depolarization of epithelial cells did not provoke a Ca²⁺ signal, and removal of external Ca²⁺ promoted rather than inhibited MLC phosphorylation. Moreover, elevation of intracellular Ca²⁺ did not induce significant MLC phosphorylation, and the myosin light chain kinase (MLCK) inhibitor ML-7 did not prevent the depolarization-induced MLC response, suggesting that MLCK is not a regulated element in this process. Instead, the Rho-Rho kinase (ROK) pathway is the key mediator because 1) depolarization stimulated Rho and induced its peripheral translocation, 2) inhibition of Rho by Clostridium difficile toxin B or C3 transferase abolished MLC phosphorylation, and 3) the ROK inhibitor Y-27632 suppressed the effect. Importantly, physiological depolarizing stimuli were able to activate the same pathway: L-alanine, the substrate of the electrogenic Na⁺-alanine cotransporter, stimulated Rho and induced Y-27632-sensitive MLC phosphorylation in a Na⁺-dependent manner. Together, our results define a novel mode of the regulation of MLC phosphorylation in epithelial cells, which is depolarization triggered and Rho-ROK-mediated but Ca²⁺ signal independent. This pathway may be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and thereby regulate paracellular transport. membrane potential; Na⁺-alanine cotransport; epithelium; phosphatidylinositol 3-kinase; LLC-PK₁ cells     

Multiple eicosanoid-activated nonselective cation channels regulate B-lymphocyte adhesion to integrin ligands

Arachidonic acid (AA) is a substrate for a variety of proinflammatory mediators, which are generated by cyclooxygenases (COXs), lipoxygenases (LOXs), and cytochrome P-450 (CYP450) enzymes. COX (e.g., PGs and prostacyclins) and LOX (e.g., leukotrienes) products have well-established proinflammatory roles; however, little is known about the functions of CYP450 products in leukocytes. We previously found that mechanical strain generated by subjecting lymphocytes to hypotonic challenge triggered AA production and that two CYP450 products of AA, 5,6-epoxyeicosatrienoic acid (5,6-EET) and 20-hydroxyeicosatetraenoic acid (20-HETE), as well as a product of LOX, 5-(S)-hydroperoxyeicosatetrenoic acid (5-HPETE), induced Ca²⁺ entry into primary B cells. The main goal of the present studies, therefore, was to define the biophysically properties of eicosanoid-activated channels responsible for Ca²⁺ entry and the physiological consequences of activating these channels, including their role in mechanical signaling. We found that 5,6-EET, 20-HETE, and 5-HPETE each activated distinct Ca²⁺-permeant nonselective cation channels (NSCCs) in primary B cells. These NSCCs each regulate plasma membrane potential and B-cell adhesion to integrin ligands ICAM-1 and VCAM-1. Thus our data demonstrate that proinflammatory mediators produced in response to osmotic and/or physical stress play a direct role in regulating the B-cell membrane potential and their adhesion to specific ECM proteins. These results not only have important implications for understanding normal mechanisms of B-cell activation, differentiation, and trafficking but also point to novel targets for modulating the pathogenesis of B-cell-mediated inflammatory diseases. calcium; arachidonic acid; membrane potential; hypotonicity; cytochrome P-450     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.