AA-induced Ca2 Research Articles

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

SUMMARYIntestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6−/− mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Arachidonic acid-induced Ca2+ entry and migration in a neuroendocrine cancer cell line

BackgroundStore-operated Ca2+ entry (SOCE) has been implicated in the migration of some cancer cell lines. The canonical SOCE is defined as the Ca2+ entry that occurs in response to near-maximal depletion of Ca2+ within the endoplasmic reticulum. Alternatively, arachidonic acid (AA) has been shown to induce Ca2+ entry in a store-independent manner through Orai1/Orai3 hetero-multimeric channels. However, the role of this AA-induced Ca2+ entry pathway in cancer cell migration has not been adequately assessed.MethodsThe present study investigated the involvement of AA-induced Ca2+ entry in migration in BON cells, a model gastro-enteropancreatic neuroendocrine tumor (GEPNET) cell line using pharmacological and gene knockdown methods in combination with live cell fluorescence imaging and standard migration assays.ResultsWe showed that both the store-dependent and AA-induced Ca2+ entry modes could be selectively activated and that exogenous administration of AA resulted in Ca2+ entry that was pharmacologically distinct from SOCE. Also, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-induced Ca2+ entry channel required the expression of Orai3 as well as Orai1. Moreover, we showed that AA treatment enhanced the migration of BON cells and that this migration could be abrogated by selective inhibition of the AA-induced Ca2+ entry.ConclusionsTaken together, these data revealed that an alternative Orai3-dependent Ca2+ entry pathway is an important signal for GEPNET cell migration.

Arachidonic acid triggers [Ca2+]i increases in rat round spermatids by a likely GPR activation, ERK signalling and ER/acidic compartments Ca2+ release.

Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca2+ from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+ using fluorescent Ca2+ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+ release from intracellular Ca2+ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.

Arachidonic acid-evoked Ca2 + signals promote nitric oxide release and proliferation in human endothelial colony forming cells

Arachidonic acid (AA) stimulates endothelial cell (EC) proliferation through an increase in intracellular Ca2+ concentration ([Ca2+]i), that, in turn, promotes nitric oxide (NO) release. AA-evoked Ca2+ signals are mainly mediated by Transient Receptor Potential Vanilloid 4 (TRPV4) channels. Circulating endothelial colony forming cells (ECFCs) represent the only established precursors of ECs. In the present study, we, therefore, sought to elucidate whether AA promotes human ECFC (hECFC) proliferation through an increase in [Ca2+]i and the following activation of the endothelial NO synthase (eNOS). AA induced a dose-dependent [Ca2+]i raise that was mimicked by its non-metabolizable analogue eicosatetraynoic acid. AA-evoked Ca2+ signals required both intracellular Ca2+ release and external Ca2+ inflow. AA-induced Ca2+ release was mediated by inositol-1,4,5-trisphosphate receptors from the endoplasmic reticulum and by two pore channel 1 from the acidic stores of the endolysosomal system. AA-evoked Ca2+ entry was, in turn, mediated by TRPV4, while it did not involve store-operated Ca2+ entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca2+]i and by inhibiting eNOS activity with NG-nitro-l-arginine methyl ester (l-NAME). Finally, AA per se did not stimulate hECFC growth, but potentiated growth factors-induced hECFC proliferation in a Ca2+- and NO-dependent manner. Therefore, AA-evoked Ca2+ signals emerge as an additional target to prevent cancer vascularisation, which may be sustained by ECFC recruitment.

Protective effect of low dose gadolinium chloride against isoproterenol-induced myocardial injury in rat.

Acute myocardial injury remains a leading cause of morbidity and mortality worldwide, and large amount of released arachidonic acid (AA) is found to be related to cardiomyocyte apoptosis and necrosis. Previous study suggested that GdCl3 completely abolished AA-induced Ca(2+) response. Thus, this study aims to investigate possible cardioprotection effect of GdCl3 on isoproterenol (ISO)-induced myocardial injury and its underlying mechanism(s). Rats that were randomly allocated to five groups: control, GdCl3, ISO, ISO + GdCl3, and ISO + verapamil. Serum levels of AA and cardiac markers, infarct area, and cell apoptosis in heart were measured by ELISA assay, TTC and TUNEL staining, respectively. Chemical interaction between AA and GdCl3 was evaluated by mass and UV spectrometry. The expressions and translocations of death receptor related molecules into lipid rafts were detected in neonatal rat ventricular myocytes by Western blots. Compared with ISO-administered rats, GdCl3 significantly ameliorated the myocardium injury, demonstrated by restoring serum cardiac troponin I, lactate dehydrogenase, creatine kinase MB and AA to near normal levels, and decreasing infarct area and cell apoptosis. In addition, an activation of AA-Fas pathway was found in ISO-induced myocardial injury, which was abrogated by GdCl3. Furthermore, AA induced cell apoptosis through clustering and activating death receptor related molecules TNFR1, Fas and FADD in lipid rafts, a process significantly prevented by the pretreatment with GdCl3. Finally, GdCl3 at the molar ratio of 1/3 (GdCl3/AA) was mostly effective in abolishing AA-induced Ca(2+) response and cell apoptosis, because an obvious change in the chemical identity of AA was obtained by GdCl3 according to this molar ratio. In conclusion, this study demonstrates for the first time that GdCl3 protects myocardium against ISO-induced cell apoptosis through, at least partly, serving as a scavenger of AA, therefore abolishing its downstream activation of the death receptor regulated apoptosis pathway.

Possible mechanisms underlying the biphasic regulatory effects of arachidonic acid on Ca2 + signaling in HEK293 cells

Arachidonic acid (AA), an endogenous lipid signal molecule released from membrane upon cell activation, modulates intracellular Ca2+ ([Ca2+]i) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca2+]i and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3μM, reduced the PM fluidity by activating PKCα and PKCβII translocation to PM and also the ER fluidity directly. In accordance, 3μM AA did not impact the basal [Ca2+]i but significantly suppressed the thapsigargin-induced Ca2+ release and Ca2+ influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCβ using shRNA significantly attenuated the inhibitory effects of 3μM AA on PM fluidity and agonist-induced Ca2+ signal. However, AA at high concentration, 30μM, caused robust release and entry of Ca2+ accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKCβI and PKCβII redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30μM AA-induced Ca2+ influx by 45%, Gd3+ at concentration of 10μM could completely abolish both release and entry of Ca2+ induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca2+ signal. Therefore, the study herein demonstrates that a lowered PM fluidity by PKC activation and a direct ER stabilization contribute significantly for AA downregulation of [Ca2+]i response, while Gd3+-sensitive ‘pores’ in PM/ER play an important role in AA-induced Ca2+ signal in HEK293 cells.

Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A

Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.

Arachidonic Acid–Induced Ca2+ Entry Is Involved in Early Steps of Tumor Angiogenesis

Growth factor-induced intracellular calcium signals in endothelial cells regulate cytosolic and nuclear events involved in the angiogenic process. Among the intracellular messengers released after proangiogenic stimulation, arachidonic acid (AA) plays a key role and its effects are strictly related to calcium homeostasis and cell proliferation. Here, we studied AA-induced intracellular calcium signals in endothelial cells derived from human breast carcinomas (B-TEC). AA promotes B-TEC proliferation and organization of vessel-like structures in vitro. The effect is directly mediated by the fatty acid without a significant contribution of its metabolites. AA induces Ca(2+)(i) signals in the entire capillary-like structure during the early phases of tubulogenesis in vitro. No such responses are detectable in B-TECs organized in more structured tubules. In B-TECs growing in monolayer, AA induces two different signals: a Ca(2+)(i) increase due to Ca(2+) entry and an inhibition of store-dependent Ca(2+) entry induced by thapsigargin or ATP. An inhibitor of Ca(2+) entry and angiogenesis, carboxyamidotriazole, significantly and specifically decreases AA-induced B-TEC tubulogenesis, as well as AA-induced Ca(2+) signals in B-TECs. We conclude that (a) AA-activated Ca(2+) entry is associated with the progression through the early phases of angiogenesis, mainly involving proliferation and tubulogenesis, and it is down-regulated during the reorganization of tumor-derived endothelial cells in capillary-like structures; and (b) inhibition of AA-induced Ca(2+) entry may contribute to the antiangiogenic action of carboxyamidotriazole.

Antiplatelet effect of sanguinarine is correlated to calcium mobilization, thromboxane and cAMP production

Sanguinarine is a plant alkaloid present in the root of Sanguinaria canadensis and Poppy fumaria species. Sanguinarine has been used as an antiseptic mouth rinse and a toothpaste additive to reduce dental plaque and gingival inflammation. In this study, we investigated the antiplatelet effects of sanguinarine, aiming to extend its potential pharmacological applications. Sanguinarine inhibited platelet aggregation induced by arachidonic acid (AA), collagen, U46619 and sub-threshold concentration of thrombin (0.05 U/ml) with IC 50 concentrations of 8.3, 7.7, 8.6 and 4.4 μM, respectively. Sanguinarine (5–10 μM) inhibited 10–31% of platelet TXB 2 production, but not platelet aggregation induced by higher concentration of thrombin (0.1 U/ml). SQ29548, a thromboxane receptor antagonist, inhibited the AA-induced platelet aggregation but not TXB 2 production. Sanguinarine suppressed cyclooxygenase-1 (COX-1) activity (IC 50 = 28 μM), whereas its effect on COX-2 activity was minimal. Sanguinarine (8, 10 μM) further inhibited the AA-induced Ca 2+ mobilization by 27–62%. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the inhibitory effect of sanguinarine toward AA-induced platelet Ca 2+ mobilization and aggregation. These results suggest that sanguinarine is a potent antiplatelet agent, which activates adenylate cyclase, inhibits platelet Ca 2+ mobilization, TXB 2 production as well as suppresses COX-1 enzyme activity. Sanguinarine may have therapeutic potential for treatment of cardiovascular diseases related to platelet aggregation.

Defect in Regulation of Ca2+ Movement in Platelets from Patients with Systemic Lupus erythematosus

The differences in the intracellular Ca²⁺ responses to hormones in platelets from systemic lupus erythematosus (SLE) patients compared to normal humans have not been explored. This study examined the Ca²⁺ signaling and density of platelets in normal, inactive and active SLE patients. The platelet number per µl 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 Ca²⁺ levels ([Ca²⁺]_i) in response to stimulation of four endogenous Ca²⁺ mobilizing hormones, 100 µM arachidonic acid (AA), 10 µM ADP, 10 nM platelet activation factor (PAF) and 1 µM thrombin, were investigated using the Ca²⁺-sensitive fluorescent dye, fura-2. The AA-induced [Ca²⁺]_i rises in normal and inactive groups were similar. In contrast, the AA-induced [Ca²⁺]_i rises in the active SLE group were significantly smaller than in the normal and inactive groups. The defect in the AA-induced [Ca²⁺]_i rises in active SLE groups appears to be caused by defective Ca²⁺ influx and Ca²⁺ releasing pathways because the AA-induced responses were not altered by removal of extracellular Ca²⁺, whereas the AA-induced responses in normal and inactive SLE groups were reduced by removal of extracellular Ca²⁺, and the AA-induced Ca²⁺ release was smaller in the active SLE group. PAF, ADP and thrombin all induced [Ca²⁺]_i rises in the three groups, but no significant differences were found among the three groups. Together, the results indicate that cell density and Ca²⁺ 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.

Direct coupling between arachidonic acid-induced Ca 2+ release and Ca 2+ entry in HEK293 cells

Arachidonic acid (AA) modulates intracellular Ca 2+ signaling via Ca 2+ release or/and Ca 2+ entry. However, the mechanism underlies either process is unknown; nor is it clear as to whether the two processes are mechanistically linked. By using Fura2/AM, we found that AA induced mobilization of internal Ca 2+ store and an increment in Ca 2+, Mn 2+ and Ba 2+ influx in HEK293 cells. The AA-mediated Ca 2+ signaling was not due to AA metabolites, and insensitive to capacitative Ca 2+ entry inhibitors. Interestingly, isotetrandrine and Gd 3+ inhibited both AA-induced Ca 2+ release and Ca 2+ entry in a concentration-dependent manner without affecting Ca 2+ discharge caused by carbachol, caffeine, or thapsigargin. Additionally, similar pattern of inhibition was observed with tetracaine treatment. More importantly, the three compounds exhibited almost equal potent inhibition of AA-initiated Ca 2+ release as well as Ca 2+ influx. Therefore, this study, for the first time, provides evidence for a direct coupling between AA-mediated Ca 2+ release and Ca 2+ entry.

Arachidonic acid regulates two Ca 2+ entry pathways via nitric oxide

Several regulated Ca 2+ entry pathways have been identified, with capacitative Ca 2+ entry (CCE) being the most characterized. In the present study, we examined Ca 2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca 2+ release from intracellular stores, and increased Ca 2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca 2+ entry when CCE was blocked by gadolinium (Gd 3+). AA-induced Ca 2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca 2+ release and Ca 2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca 2+, and to activate a second Ca 2+ entry pathway that is dependent on depletion of Ca 2+ from ryanodine-sensitive stores.

Activation of leukotriene synthesis in human neutrophils by exogenous arachidonic acid: inhibition by adenosine A(2a) receptor agonists and crucial role of autocrine activation by leukotriene B(4).

We report here that the apparent inability of isolated human polymorphonuclear leukocytes (PMNs) to efficiently transform arachidonic acid (AA) is the consequence of A(2a) receptor engagement by endogenous adenosine accumulating in incubation media. Indeed, when adenosine is eliminated from PMN suspensions by the addition of adenosine deaminase, or when cells are incubated with adenosine A(2a) receptor antagonists, important quantities (40-80 pmol/10(6) cells) of 5-lipoxygenase products are synthesized by PMN incubated with 1 to 5 microM exogenous AA. The selective A(2a) receptor agonist CGS21680 was a very potent inhibitor of the AA-induced leukotriene (LT) synthesis, showing an IC(50) of approximately 1 nM. The mechanism of AA-induced stimulation of LT synthesis observed in the absence of extracellular adenosine was investigated. In adenosine deaminase-treated PMN, exogenous AA induced Ca(2+) mobilization and the translocation of 5-lipoxygenase to nuclear structures. A time lag of 20 to 60 s (variable between PMN preparations) was observed consistently between the addition of AA and the elevation of intracellular Ca(2+) concentration (and LT synthesis), indicating that AA itself did not trigger the Ca(2+) mobilization in PMN. This AA-induced Ca(2+) mobilization, as well as the corresponding 5-lipoxygenase translocation and stimulation of LT synthesis, was blocked efficiently by the LT synthesis inhibitor MK0591, the LTB(4) receptor antagonists CP105696 and LY223982, and the LTA(4) hydrolase inhibitor SC57461A. These data demonstrate that AA is a highly potent and effective activator of LT synthesis and acts through a mechanism that requires an autocrine stimulatory loop by LTB(4).

Differential Regulation of Phospholipase A2(PLA2)-dependent Ca2+ Signaling in Smooth Muscle by cAMP- and cGMP-dependent Protein Kinases: INHIBITORY PHOSPHORYLATION OF PLA2 BY CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES

Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C-beta (PLC-beta) activity and inositol 1,4,5-trisphosphate-dependent Ca2+ release in vascular and visceral smooth muscle. In smooth muscle of the intestinal longitudinal layer, however, the initial steps in Ca2+ mobilization involve activation of cytosolic PLA2 (cPLA2) and arachidonic acid (AA)-dependent stimulation of Ca2+ influx. The present study examined whether cAMP- and cGMP-dependent protein kinases are capable of regulating these processes also. Agents that activated cAMP-dependent protein kinase (5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (Sp-isomer) and isoproterenol), cGMP-dependent protein kinase (8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate and Na nitroprusside), or both kinases (vasoactive intestinal peptide and isoproterenol >1 microM) induced phosphorylation of cPLA2 and inhibition of agonist-stimulated cPLA2 activity. Phosphorylation and inhibition of cPLA2 activity by cAMP- and cGMP-dependent protein kinases were blocked by the corresponding selective inhibitors (cAMP-dependent protein kinase, N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide hydrochloride (H-89) and myristoylated protein kinase inhibitor () amide; cGMP-dependent protein kinase, (8R,9S, 11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c, d, e)-trinden-1-one (KT-5823)). In contrast, AA-stimulated Ca2+ influx was inhibited by agents that activated cGMP-dependent protein kinase only; the inhibition was selectively blocked by KT-5823. The study provides the first evidence of inhibitory phosphorylation of cPLA2 in vivo by cAMP- and cGMP-dependent protein kinases. Inhibition of cPLA2 activity and AA-induced Ca2+ influx partly account for the ability of cAMP-dependent protein kinase and/or cGMP-dependent protein kinase to cause relaxation. Their importance resides in their location at the inception of the Ca2+ signaling cascade.

Arachidonic acid-induced hormone release in somatotropes: involvement of calcium.

Arachidonic acid (AA) has been implicated in signaling actions in several cell types including endocrine cells. In the present study, we investigated the effect of exogenous AA on GH release from dispersed pituitary cells and tried to elucidate the mechanism involved in this process. We show that AA stimulates GH release in a dose- and extracellular calcium-dependent manner. The effects of AA on cytosolic calcium concentration ([Ca2+]i) were studied using dual-emission microspectrofluorimetry in identified somatotropes. AA (1 microM) induced an increase in intracellular calcium concentration ([Ca2+]i) by stimulating Ca2+ influx through dihydropyridine-sensitive, voltage-dependent calcium channels. In these cells, the effects of AA were only reduced by the inhibition of protein kinase C (PKC) activity, suggesting that the fatty acid may act by both PKC-dependent and PKC-independent pathways. In order to determine whether AA metabolites were involved in the effects attributed to AA, and, if so, which ones, we inhibited the three arachidonate metabolic pathways: cyclo-oxygenase by indomethacin (50 microM), lipoxygenase by nordihydroguaiaretic acid (NGDA, 50 microM), and epoxygenase by 5,8,11, 14-eicosatetraynoic acid (ETYA, 10 microM). NGDA and ETYA reduced the effects of AA on GH release (50 and 74%, respectively) and inhibited the [Ca2+]i response, whereas indomethacin slightly potentiated both AA-induced GH release and [Ca2+]i increase. As these results suggested that lipoxygenase metabolites may be responsible for AA-induced Ca2+ influx and GH release, we tested the effects of 5-, 12- and 15-hydroperoxyeicosatetraenoic acids (5-, 12- and 15-HpETE) on [Ca2+]i and GH release. They all stimulated calcium influx and GH release in a dose-dependent manner, 12-HpETE being more potent than 5- and 15-HpETE. We conclude that lipoxygenase metabolites of arachidonic acid, particularly 12-HpETE, may be involved in the GH secretion mechanism, probably by facilitating Ca2+ influx via L-type Ca2+ channels.

Arachidonic acid increases cytosolic calcium and stimulates hormone release in rat lactotrophs

Arachidonic acid (AA) released from membrane phospholipids after activation of surface receptors causes cellular signaling actions in neurons and endocrine cells, including stimulation of prolactin (PRL) release from dissociated rat pituitary cells and clonal cells of the GH3 pituitary tumor line. In the present study, we investigated the effect of exogenous AA on PRL release from dispersed pituitary cells and tried to elucidate the mechanism involved in this process. The effects of AA on cytosolic Ca2+ concentration ([Ca2+]i) were studied using dual-emission microspectrofluorometry in identification lactotrophs and on PRL release in dispersed pituitary cell populations. AA had a dose-dependent effect on [Ca2+]i. At 1 microM, the Ca2+ increase was biphasic: a mobilization of intracellular Ca2+ from intracellular stores was followed by stimulation of Ca2+ influx. For lower concentrations (10 and 100 nM), only the stimulation of Ca2+ influx was observed. AA-induced Ca2+ influx and PRL release were not due to the stimulation of a phorbol 12-myristate 13-acetate-sensitive protein kinase C. In the same way, AA-stimulated PRL release and intracellular Ca2+ increase were independent of intracellular thapsigargin-sensitive Ca2+ pools. Furthermore, blockade of Ca2+ channels suppressed AA-induced PRL release. We hypothesize that Ca2+ influx plays a major role in AA-induced PRL release.

Arachidonic acid is functioning as a second messenger in activating the Ca2+ entry process on H1-histaminoceptor stimulation in DDT1 MF-2 cells

This study was carried out to identify the cellular component activating the histamine-stimulated Ca2+ entry in vas-deferens-derived DDT1 MF-2 cells. H1-histaminoceptor stimulation resulted in a rise in intracellular Ca2+ concentration, caused by Ca2+ release from inositol phosphate-sensitive Ca2+ stores and Ca2+ entry from the extracellular space, accompanied by a transient Ca(2+)-activated outward K+ current. The histamine-evoked K+ current was still observed after preventing inositol phosphate-induced Ca2+ mobilization by intracellularly applied heparin. This current was activated by Ca2+ entry from the extracellular space, because it was abolished in the presence of the Ca(2+)-channel blocker La3+ or under Ca(2+)-free conditions. H1-histaminoceptor-activated Ca2+ entry was also observed in the presence of intracellularly applied Ins(1,4,5)P3 and Ins(1,3,4,5)P4, depleting their respective Ca2+ stores and pre-activating the inositol phosphate-regulated Ca2+ entry. Thus the ability of histamine to activate Ca2+ entry independently of Ca2+ mobilization and the formation of inositol phosphates suggests that another component is involved to initiate the Ca(2+)-entry process. It was observed that H1-histaminoceptor stimulation resulted in a pronounced release of arachidonic acid (AA) in DDT1 MF-2 cells. Exogenously applied AA induced a concentration-dependent increase in internal Ca2+ due to activation of Ca2+ entry from the extracellular space. Slow inactivation of the AA-sensitive Ca2+ channels is suggested by the slow decline in Ca2+ entry. In accord, the histamine-induced Ca2+ entry was not observed with AA-pre-activated Ca2+ channels. Inhibition of the lipoxygenase and cyclo-oxygenase pathway did not affect the AA-induced Ca2+ and the concomitant K+ current were decreased in the presence of AA and caused by Ca2+ mobilization from internal stores. Blocking this internal Ca2+ release by heparin, in the presence of AA, resulted in abolition of the histamine-induced Ca(2+)-regulated K+ current. These observations show that AA, released on H1-histaminoceptor stimulation in DDT1 MF-2 cells, is functioning as a second messenger to activate plasma-membrane Ca2+ channels promoting Ca2+ entry from the extracellular space.

ω-Conotoxin inhibits glucose- and arachidonic acid-induced rises in intracellular [Ca 2+] in rat pancreatic islet β-cells

Earlier studies suggest that the accumulation of non-esterified arachidonic acid (AA) in islets following stimulation with glucose participates in the glucose-induced secretion of insulin. A possible role for AA might include the facilitation of Ca 2+ influx into islet β-cells. Recently, we demonstrated that AA induces Ca 2+ influx into purified rat pancreatic islet β-cells, prepared by fluorescence-activated cell sorting (FACS). This effect was abolished in the presence of the Ca 2+-chelator EGTA, but was only partially reduced by the dihydropyridine (DHP) L-type Ca 2+-channel blocker, nifedipine. This raised the possibility that DHP- insensitive Ca 2+ entry mechanisms may exist in pancreatic β-cells, in addition to the known DHP-sensitive L-type Ca 2+ channels. Here we report that ω-conotoxin (CTX), which blocks ω-type Ca 2+-channels, inhibits AA-induced Ca 2+ influx by a magnitude similar to that of nifedipine and that the combination of ω-CTX and nifedipine results in a nearly additive decrement in AA-induced increases in β-cell cytosolic [Ca 2+]. We further demonstrate that bovine serum albumin, which complexes free AA and prevents AA-induced increases in cytosolic [Ca 2+], also inhibits the glucose-induced increase in β-cell [Ca 2+]. These results suggest that rat pancreatic FACS-purified islet β-cells express ω-type (DHP- insensitive) Ca 2+-channels, in addition to DHP-sensitive Ca 2+-channels. They further suggest that the glucose-induced accumulation of non-esterified AA in the membranes of β-cells serves to amplify glucose-mediated Ca 2+ influx into the β-cells.

Arachidonic acid and its metabolites increase Cai in cultured rat oligodendrocytes

Fluorescence measurements of intracellular calcium (Cai) were made on cultured rat spinal cord oligodendrocytes (OLGs) using the dye fura-2. Exposure of OLGs to arachidonic acid (AA) (5-50 microM) elicited a concentration-dependent increase in Cai that was derived mainly from extracellular Ca2+. AA at 50 microM also released Ca2+ from intracellular stores. The response to AA was not decreased by nifedipine or by inhibition of Na(+)-Ca2+ exchange. AA-induced Ca2+ influx pathway was permeable to Mn2+ and Co2+ but not to Ba2+ and was not markedly influenced by depolarization, suggesting that AA activates a voltage-independent, not strictly selective, Ca2+ channel. The Cai response to AA was partially attenuated in the presence of indomethacin, indicating that the Cai response was mediated in part by cyclooxygenase products of AA. However, the AA-induced Cai response far exceeded that induced by prostaglandins and was mimicked by linoleic acid. We conclude that AA modulates Cai of OLGs via two mechanisms: 1) indirectly via cyclooxygenase pathway and 2) directly via membrane lipid-protein interaction.

Regulation of Arachidonic Acid-Dependent Ca++ Influx in Human Platelets

Quin2 was used to study the rise in cytoplasmic free calcium ([Ca++]i) and the role of prostaglandin (PG) endoperoxides/thromboxane A2 (TxA2), reduced glutathione (GSH), ADP and the glycoprotein (GP) IIb-IIIa complex in mediating [Ca++]i rise during arachidonic acid (AA)-induced platelet aggregation. Ca++ mobilization, mostly due to an influx across the plasma membrane, is completely inhibited by aspirin and persists after selective blockade of TxA2 synthase by dazoxiben. GSH total depletion causes a complete aggregation block and 90% inhibition of the transient: U-46619, a stable analog of cyclic endoperoxide PGH2, stimulates [Ca++]i transient in aspirin-treated or in GSH-depleted platelets. ADP-scavengers, ATP (which competes for the ADP receptor), and monoclonal antibodies against the GP IIb-IIIa complex reduce AA-induced Ca++ influx. Therefore, PG endoperoxides alone or a PGH2/TxA2 mimetic stimulate Ca++ influx. Synthesis of PGH2 and TxA2 depends on the availability of GSH, which acts as the reducing cofactor for the PG-peroxidase activity. ADP and GP IIb-IIIa are regulating factors of AA-mediated Ca++ influx during platelet activation.