Inhibitory Effect Of Arachidonic Acid Research Articles

Arachidonic acid induces ER stress and apoptosis in HT-29 human colon cancer cells

ABSTRACT Polyunsaturated fatty acids (PUFAs) have important functions in biological systems. The beneficial effects of dietary PUFAs against inflammatory diseases, cardiovascular diseases, and metabolic disorders have been shown. Studies using cancer cells have presented the anti-tumorigenic effects of docosahexaenoic acid (DHA), an n-3 PUFA, while arachidonic acid (AA), an n-6 PUFA, has been shown to elicit both pro- and anti-tumorigenic effects. In the current study, the anti-tumorigenic effects of AA were evaluated in HT-29 human colon cancer cells. Upon adding AA in the media, more than 90% of HT-29 cells died, while the MCF7 cells showed good proliferation. AA inhibited the expression of SREBP-1 and its target genes that encode enzymes involved in fatty acid synthesis. As HT-29 cells contained lower basal levels of fatty acid synthase, a target gene of SREBP-1, than that in MCF7 cells, the inhibitory effects of AA on the fatty acid synthase levels in HT-29 cells were much stronger than those in MCF-7 cells. When oleic acid (OA), a monounsaturated fatty acid that can be synthesized endogenously, was added along with AA, the HT-29 cells were able to proliferate. These results suggested that HT-29 cells could not synthesize enough fatty acids for cell division in the presence of AA because of the suppression of lipogenesis. HT-29 cells may incorporate more AA into their membrane phospholipids to proliferate, which resulted in ER stress, thereby inducing apoptosis. AA could be used as an anti-tumorigenic agent against cancer cells in which the basal fatty acid synthase levels are low.

Long-chain polyunsaturated fatty acids amend palmitate-induced inflammation and insulin resistance in mouse C2C12 myotubes.

Intramuscular lipid accumulation results in inflammation, which is correlated with impaired insulin action in the skeletal muscle, an important organ for glucose uptake in the body. In this study, we explored the effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), arachidonic acid (AA), and long-chain polyunsaturated fatty acids (PUFAs) on palmitic acid (PA)-induced inflammatory responses and insulin resistance in C2C12 myotubes. The mRNA expression of the pro-inflammatory cytokines interleukin-6 and tumor necrosis factor-α in PA-treated myotubes was suppressed by these three test long-chain PUFAs. Moreover, the addition of long-chain PUFAs decreased PA-induced insulin resistance as evidenced by increases in phosphorylated AKT and glucose uptake. In PA-treated myotubes, long-chain PUFAs improved glucose transporter 4 expression, basal glucose uptake without insulin, and the AMP-activated protein kinase pathway. Of note, the long-chain PUFAs obstructed the effects of PA on the activation of extracellular-signal-regulated kinase and protein kinase C-θ as well as nuclear factor-κB (NF-κB) and activator protein-1. The inhibitory effect of AA but not of DHA and EPA on PA-induced inflammation and impaired insulin action was cancelled in C2C12 myotubes transfected with a constitutively active mutant IκB kinase-β plasmid. These data suggest that long-chain PUFAs may be useful in the management of PA-induced inflammation and insulin resistance in myotubes. In addition to the NF-κB pathway, other mechanisms are involved in the health benefits of DHA and EPA in PA-treated myotubes.

Cyp2c44 Epoxygenase Is Essential for Preventing the Renal Sodium Absorption During Increasing Dietary Potassium Intake

The aim of this study is to test whether the Cyp2c44 epoxygenase-dependent metabolism of arachidonic acid prevents the hypertensive effect of a high K (HK) intake by inhibiting the epithelial sodium channel (ENaC) activity. A HK intake elevated Cyp2c44 mRNA expression and 11,12-epoxyeicosatrienoic acid levels in the cortical collecting duct in Cyp2c44(+/+) mice (wild-type [wt]). However, an HK intake failed to increase 11,12-epoxyeicosatrienoic acid formation in the cortical collecting ducts of Cyp2c44(-/-) mice. Moreover, increasing K intake enhanced arachidonic acid-induced inhibition of ENaC in the wt but not in Cyp2c44(-/-) mice. In contrast, 11,12-epoxyeicosatrienoic acid, a Cyp2c44 metabolite, inhibited ENaC in the wt and Cyp2c44(-/-) mice. The notion that Cyp2c44 is the epoxygenase responsible for mediating the inhibitory effects of arachidonic acid on ENaC is further suggested by the observation that inhibiting Cyp-epoxygenase increased the whole-cell Na currents in principal cells of wt but not in Cyp2c44(-/-) mice. Feeding mice with an HK diet raised the systemic blood pressures of Cyp2c44(-/-) mice but was without an effect on wt mice. Moreover, application of amiloride abolished the HK-induced hypertension in Cyp2c44(-/-) mice. The HK-induced hypertension of Cyp2c44(-/-) mice was accompanied by decreasing 24-hour urinary Na excretion and increasing the plasma Na concentration, and the effects were absent in wt mice. In contrast, disruption of the Cyp2c44 gene did not alter K excretion. We conclude that Cyp2c44 epoxygenase mediates the inhibitory effect of arachidonic acid on ENaC and that Cyp2c44 functions as an HK-inducible antihypertensive enzyme responsible for inhibiting ENaC activity and Na absorption in the aldosterone-sensitive distal nephron.

Arachidonic acid inhibits Na+–K+–ATPase via cytochrome P-450, lipoxygenase and protein kinase C-dependent pathways in sheep pulmonary artery

The purpose of the study was to examine whether arachidonic acid inhibits vascular Na+–K+–ATPase in pulmonary vasculature and if so, what are the mechanisms involved. Functional Na+–K+–ATPase activity was studied in terms of K+-induced relaxation in sheep pulmonary arterial rings contracted with K+-free solution and 5-HT. Arachidonic acid (10–100μM) caused concentration-dependent inhibition of KCl-induced relaxations and also increased basal arterial tone. Cytochrome P-450 inhibitor, 17-octadecynoic acid (17-ODYA) completely reversed the arachidonic acid (30μM)-induced inhibition of KCl relaxation. Further, in the presence of HET0016, a selective blocker of 20-hydroxyeicosatetraenoic acid (20-HETE), arachidonic acid-induced inhibition of KCl relaxation was not evident. Accordingly, 20-HETE, a cytochrome P-450 metabolite of arachidonic acid, also significantly attenuated KCl-induced relaxations. Norhydihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, however, partially restored the relaxation to K+, impaired in the presence of arachidonic acid (30μM). On the other hand, cyclooxygenase inhibitor indomethacin failed to reverse the inhibitory effect of arachidonic acid on KCl-induced relaxation. Staurosporin, a protein kinase C inhibitor, completely reversed the inhibitory effect of arachidonic acid and 20-HETE on K+-induced relaxation. In conclusion, the results suggest that 20-HETE, a cytochrome P-450 metabolite of arachidonic acid has a predominant role in the inhibition of functional Na+–K+–ATPase activity in the sheep pulmonary artery, while the lipooxygenase pathway has a secondary role. It is also evident that protein kinase C is involved in the inhibition of Na+–K+–ATPase by arachidonic acid/20-HETE in sheep pulmonary artery.

Inhibitory effect of arachidonic acid on venom acetylcholinesterase

The venom of krait (Bungarus sindanus), an Elapidae snake, contains high level of acetylcholinesterase (AChE) activity. The effect of arachidonic acid on krait (Bungarus sindanus) venom AChE (acetylcholine hydrolase, EC 3.1.1.7) was studied using kinetic methods. This study evaluated for the first time whether arachidonic acid acts as potential AChE inhibitor of krait venom. Kinetic studies, using Line-Weaver Burk and Dixon plots, indicated that the inhibition produced by arachidonic acid was non-competitive, i.e., both km and V max fell with increase in the concentration of arachidonic acid. The concentration of arachidonic acid required to inhibit 50% of enzymatic activity was found to be 1.9 µM while the inhibitory constant (Ki ) was 1.4 µM. The present results suggest that essential fatty acid (arachidonic acid) may also be used as an inhibitor of snake venom AChE.

Identification of a Kv4 channel hydrophobic binding pocket for the inhibitory effects of polyunsaturated fatty acids

Polyunsaturated fatty acids such as arachidonic acid (AA) inhibit Kv4 potassium channel function and alter neuronal excitability. We tested the hypothesis that the inhibitory effects of AA on Kv4.2/KChIP channels require access to a hydrophobic binding site within a membrane-embedded region of the channel. We prepared a homology model of Kv4.2 and used a molecular docking approach to identify residues that may interact with AA. The importance of each residue was tested by site-directed mutagenesis and two-microelectrode voltage clamp electrophysiology in Xenopus oocytes. Wild-type currents were 30% inhibited by a 5 min exposure to 10 μM AA, applied externally. We identified several mutations that significantly impaired AA inhibition. We confirmed the molecular disruption of the AA binding pocket by simulating the mutagenic substitution in our Kv4.2 molecular model. Mutations introduced to residues located near but not within the putative binding pocket did not disrupt the fatty acid inhibition of Kv4.2 current, indicating the specificity of the residues which comprise the binding pocket. Our results suggest that AA may inhibit Kv4 channel function by interfering with the function of structural domains which are not in the permeation pathway. Funded by a grant from the Jeffress Foundation (LMB) and an APS Summer Undergraduate Fellowship (RH).

Stimulation of A2a adenosine receptor abolishes the inhibitory effect of arachidonic acid on the basolateral 50-pS K channel in the thick ascending limb

The basolateral 50-pS K channels are stimulated by a cAMP-dependent pathway and inhibited by cytochrome P-450-omega-hydroxylase-dependent metabolism of arachidonic acid (AA) in the rat thick ascending limb (TAL). We now used the patch-clamp technique to examine whether stimulation of adenosine A(₂a) receptor modulates the inhibitory effect of AA on the basolateral 50-pS K channels in the medullary TAL. Stimulation of adenosine A(₂a) receptor with CGS-21680 or inhibition of phospholipase A₂ (PLA₂) with AACOCF3 increased the 50-pS K channel activity in the TAL. Western blot demonstrated that application of CGS-21680 decreased the phosphorylation of PLA(2) at serine residue 505, an indication of inhibiting PLA₂ activity. In the presence of CGS-21680, inhibition of PLA₂ had no further effect on the basolateral 50-pS K channels. The possibility that CGS-21680-induced stimulation of the basolateral 50-pS K channels was partially achieved by inhibition of PLA₂ in the TAL was also supported by the observation that CGS-21680 had no additional effect in the presence of AACOCF3. Moreover, stimulation of adenosine A(₂a) receptor with CGS-21680 also abolished the inhibitory effect of AA and 20-hydroxyeicosatetraenoic acid (20-HETE) on the 50-pS K channels. The effect of CGS-21680 on AA and 20-HETE-mediated inhibition of the 50-pS K channels was mediated by cAMP because application of membrane-permeable cAMP analog, dibutyryl-cAMP, not only increased the 50-pS K channel activity but also abolished the inhibitory effect of AA and 20-HETE. We conclude that stimulation of adenosine A(₂a) receptor increased the 50-pS K channel activity in the TAL, an effect that is achieved by suppression of PLA₂ activity and 20-HETE-induced inhibition.

High K (HK) intake enhances the inhibitory effect of Arachidonic Acid (AA) to ENaC by stimulating CYP Epoxygenase and inhibiting soluble Epoxide Hydrolase (sEH)

We used the patch‐clamp technique to study the effect of AA on ENaC in the CCD in rat on HK diet or on Na‐deficient diet (Na‐D). AA inhibits ENaC activity and is mediated by 11,12‐EET. However, the sensitivity of ENaC to AA is higher in rats on HK diet (ED‐50, 3 µM) than that on Na‐D (ED‐50, 5 µM). Moreover, HK intake significantly increased the expression of CYP2C23 and 11,12 EET content in the CCD. The inhibitory effect of 11,12‐EET to ENaC is enhanced in rats on HK diet which decreases ED‐50 of 11,12‐EET from 70 nM in rats on Na‐D to 30 nM. However, inhibiting sEH, which converts EET to DHET (a weak inhibitor of ENaC), increased significantly the sensitivity of ENaC to 11,12‐EET in the rats on Na‐D to the extent similar to that on HK. Finally, the observation that HK intake suppresses the metabolic rate of converting EET to DHET in the kidney suggests that HK intake may inhibit sEH and, consequently, enhance the sensitivity of ENaC to EET. We conclude that HK intake enhances the inhibitory effect of AA and 11,12‐EET on ENaC, increasing CYP epoxygenase activity and decreasing sEH activity are partially responsible for augmenting the inhibition of AA and 11,12‐EET, respectively.

Swelling-Activated Cl− Current in Isolated Rabbit Articular Chondrocytes: Inhibition by Arachidonic Acid

Articular chondrocytes play an important role in maintaining the structure and function of the cartilage in synovial joints, which is closely influenced by mechanical or osmotic stress. In the present study, isolated rabbit articular chondrocytes were examined during hyposmotic stress using the whole-cell patch-clamp method. When exposed to hyposmotic external solutions (approximately 5% or 32% decrease in osmolarity), isolated rabbit articular chondrocytes exhibited hyposmotic cell swelling, accompanied by the activation of the swelling-activated Cl− current (ICl,swell). ICl,swell was practically time-independent at potentials negative to +50 mV but exhibited rapid inactivation at more positive potentials. ICl,swell was potently inhibited by the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and tamoxifen, but was little affected by pimozide. ICl,swell was also found to be acutely inhibited by arachidonic acid in a concentration-dependent manner with an IC50 of 0.81 μM. The maximal effect (approximately 100% block) was obtained with 10 μM arachidonic acid. The arachidonic acid metabolites prostaglandin E2, leukotriene B4, and leukotriene D4 had no appreciable effect on ICl,swell, suggesting that the inhibitory effect of arachidonic acid did not require its metabolism. The present study thus reveals the presence of ICl,swell in rabbit articular chondrocytes that exhibits high sensitivity to direct inhibition by arachidonic acid.

Modulation of muscle rNav1.4 Na+ channel isoform by arachidonic acid and its non‐metabolized analog

Arachidonic acid (AA) and its metabolic products are important second messengers which exert many biological actions, including modulation of various ion channels. However, the blockage of muscle Na(+) channel isoforms by AA has not been examined in detail. Here, we investigated the modulating effects of AA on muscle rNa(V)1.4 isoforms expressed in human embryonic kidney 293 cells. The results revealed that AA has both activation and inhibitory effects on rNa(V)1.4 currents depending on the depolarizing potential: AA increased the rNa(V)1.4 current evoked by a depolarization of -30 or -40 mV, but significantly decreased the rNa(V)1.4 current evoked by a depolarization of membrane potential over -10 mV. At concentrations of 1-500 microM, the inhibitory effect on the rNa(V)1.4 current induced by AA was dose-dependent and reversible. In addition to modulating the amplitude of the rNa(V)1.4 current, AA significantly modulated the steady-state activation and inactivation properties of rNa(V)1.4 channels. Furthermore, treatment with AA resulted in a fairly slow recovery of the rNa(V)1.4 channel from inactivation; however, the inhibitory effect of AA was not changed by repetitive pulses or by changing frequency. The effect of AA on rNa(V)1.4 currents was completely mimicked by ETYA, the non-metabolized analog of AA. Our data demonstrated that AA, but not the metabolic products of AA, can voltage-dependent modulate rNa(V)1.4 currents.

Involvement of PPARα in the growth inhibitory effect of arachidonic acid on breast cancer cells

Epidemiological studies suggest that dietary PUFA may influence breast cancer progression. n-3 PUFA are generally known to exert antitumour effects, whereas reports relative to n-6 PUFA anti-carcinogen effects are controversial. Arachidonic acid (AA; 20:4n-6) and its metabolites have been shown to inhibit the growth of human breast cancer cell lines, even if the downstream mechanisms by which AA may influence carcinogenesis remain unresolved. We explored the molecular basis for AA influence on proliferation, signal transduction and apoptosis in two human breast cancer cell lines, MCF-7 and MDA-MB-231. In both cell lines AA inhibited cell growth in a dose-dependent manner, even if MDA-MB-231 was somewhat more growth-inhibited than MCF-7. AA decreased extracellular signal-regulated protein kinase 1/2 phosphorylation level, and positively modulated PPARgamma and PPARalpha expression, with only a slight effect against PPARbeta/delta. In addition, AA increased Bak (an apoptosis-regulating protein) expression and reduced procaspase-3 and -9 levels only in MDA-MB-231 cells, thus indicating that the growth inhibitory effect can be correlated with apoptosis induction. In both cell lines the use of a specific antagonist made it possible to establish a relationship between AA growth inhibitory effect and PPARalpha involvement. AA decreases cell proliferation most likely by inducing apoptosis in MDA-MB-231 cells, while in the MCF-7 cell line the growth inhibitory activity can be attributed to the inhibition of the signal transduction pathway involved in cell proliferation. In both cases, the results here presented suggest PPARalpha as a possible contributor to the growth inhibitory effect of AA.

Arachidonic acid inhibits basolateral K channels in the cortical collecting duct via cytochrome P-450 epoxygenase-dependent metabolic pathways

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on basolateral 18-pS K channels in the principal cell of the cortical collecting duct (CCD) of the rat kidney. Application of AA inhibited the 18-pS K channels in a dose-dependent manner and 10 microM AA caused a maximal inhibition. The effect of AA on the 18-pS K channel was specific because application of 11,14,17-eicosatrienoic acid had no effect on channel activity. Also, the inhibitory effect of AA on the 18-pS K channels was abolished by blocking cytochrome P-450 (CYP) epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) but was not affected by inhibiting CYP omega-hydroxylase or cyclooxygenase. The notion that the inhibitory effect of AA was mediated by CYP epoxygenase-dependent metabolites was further supported by the observation that application of 100 nM 11,12-epoxyeicosatrienoic acid (EET) mimicked the effect of AA and inhibited the basolateral 18-pS K channels. In contrast, addition of either 5,6-, 8,9-, or 14,15-EET failed to inhibit the 18-pS K channels. Moreover, application of 11,12-EET was still able to inhibit the 18-pS K channels in the presence of MS-PPOH. This suggests that 11,12-EET is a mediator for the AA-induced inhibition of the 18-pS K channels. We conclude that AA inhibits basolateral 18-pS K channels by a CYP epoxygenase-dependent pathway and that 11,12-EET is a mediator for the effect of AA on basolateral K channels in the CCD.

Arachidonic acid (AA) inhibits basolateral K channels in the cortical collecting duct (CCD) via cytochrome P450 (CYP) epoxygenase‐dependent metabolic pathways

We used the patch‐clamp technique to study the effect of AA on basolateral 18 pS K channels in principal cell of the CCD of the rat kidney. Application of AA inhibited the 18 pS K channels. The effect of AA on the 18 pS K channel was specific because application of 11,14,17‐eicosatrienoic acid had no effect. Also, the inhibitory effect of AA on the 18 pS K channels was abolished by blocking CYP epoxygenase with MS‐PPOH but was not affected by DDMS, an inhibitor of CYP monoxygenase. The notion that the inhibitory effect of AA was mediated by CYP epoxygenase‐dependent metabolites was also supported by the observation that application of 100 nM 11,12‐epoxyeicosatrienoic acid (EET) mimicked the effect of AA and inhibited the basolateral 18 pS K channels. In contrast, addition of either 8,9‐, or 14,15‐EET failed to inhibit the 18 pS K channels. Moreover, application of 11,12‐EET was still able to inhibit the 18 pS K channels in the presence of MS‐PPOH. This suggests that 11,12‐EET is a mediator for the AA‐induced inhibition of the 18 pS K channels. We conclude that AA inhibits basolateral 18 pS K channels in the CCD and that the effect of AA is mediated by a CYP epoxygenase‐dependent metabolite, 11,12‐EET.

Control of cyclic AMP concentration in bovine endometrial stromal cells by arachidonic acid

Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E(2) during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE(2). cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE(2) increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4beta-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE(2)-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE(2), forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE(2). The data suggest that arachidonic acid antagonizes PGE(2) signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.

Effects of arachidonic acid, docosahexaenoic acid, prostaglandin E 2 and parathyroid hormone on osteoprotegerin and RANKL secretion by MC3T3-E1 osteoblast-like cells

Bone is continuously remodeled through resorption by osteoclasts and the subsequent synthesis of the bone matrix by osteoblasts. Cell-to-cell contact between osteoblasts and osteoclast precursors is required for osteoclast formation. RANKL (receptor activator of nuclear factor-κB ligand) expressed on osteoblastic cell membranes stimulates osteoclastogenesis, while osteoprotegerin (OPG) secreted by osteoblasts inhibits osteoclastogenesis. Although polyunsaturated fatty acids (PUFAs) have been implicated in bone homeostasis, the effects thereof on OPG and RANKL secretion have not been investigated. MC3T3-E1 osteoblasts were exposed to the n−6 PUFA arachidonic acid (AA) and the n−3 PUFA docosahexaenoic acid (DHA); furthermore, the bone-active hormone parathyroid hormone (PTH) and the effects thereof were tested on OPG and RANKL secretion. Prostaglandin E 2 (PGE 2), a product of AA metabolism that was previously implicated in bone homeostasis, was included in the study. AA (5.0–20 μg/ml) inhibited OPG secretion by 25–30%, which was attenuated by pretreatment with the cyclooxygenase blocker indomethacin, suggesting that the inhibitory effect of AA on OPG could possibly be PGE 2-mediated. MC3T3-E1 cells secreted very low basal levels of RANKL, but AA stimulated RANKL secretion, thereby decreasing the OPG/RANKL ratio. DHA suppressed OPG secretion to a smaller extent than AA. This could, however, be due to endogenous PGE 2 production. No RANKL could be detected after exposing the MC3T3-E1 cells to DHA. PTH did not affect OPG secretion, but stimulated RANKL secretion. This study demonstrates that AA and PTH reduce the OPG/RANKL ratio and may increase osteoclastogenesis. DHA, however, had no significant effect on OPG or RANKL in this model.

Resistin expression in 3T3-L1 adipocytes is reduced by arachidonic acid

The resistin gene is expressed in adipocytes and encodes a protein proposed to link obesity and type 2 diabetes. Increased plasma FFA is associated with insulin resistance. We examined the effect of separate FFAs on the expression of resistin mRNA in cultured murine 3T3-L1 adipocytes. The FFAs tested did not increase resistin expression, whereas both arachidonic acid (AA) and eicosapentaenoic acid (EPA) reduced resistin mRNA levels. AA was by far the most potent FFA, reducing resistin mRNA levels to approximately 20% of control at 60-250 muM concentration. Selective inhibitors of cyclooxygenase-1 and of mitogen-activated protein kinase kinase counteracted AA-induced reduction in resistin mRNA levels. Transient overexpression of sterol-regulatory element binding protein-1a (SREBP-1a) activated the resistin promoter, but there was no reduction in the abundance of approximately 65 kDa mature SREBP-1 after AA exposure. Actinomycin D as well as cycloheximide abolished the AA-induced reduction of resistin mRNA levels, indicating dependence on de novo transcription and translation. Our data suggest that reductions in resistin mRNA levels involve a destabilization of the resistin mRNA molecule. An inhibitory effect of AA and EPA on resistin expression may explain the beneficial effect of ingesting PUFAs on insulin sensitivity.

Arachidonic Acid Inhibits Epithelial Na Channel Via Cytochrome P450 (CYP) Epoxygenase-dependent Metabolic Pathways

We used the patch-clamp technique to study the effect of arachidonic acid (AA) on epithelial Na channels (ENaC) in the rat cortical collecting duct (CCD). Application of 10 μM AA decreased the ENaC activity defined by NPo from 1.0 to 0.1. The dose–response curve of the AA effect on ENaC shows that 2 μM AA inhibited the ENaC activity by 50%. The effect of AA on ENaC is specific because neither 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolized analogue of AA, nor 11,14,17-eicosatrienoic acid mimicked the inhibitory effect of AA on ENaC. Moreover, inhibition of either cyclooxygenase (COX) with indomethacin or cytochrome P450 (CYP) ω-hydroxylation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) failed to abolish the effect of AA on ENaC. In contrast, the inhibitory effect of AA on ENaC was absent in the presence of N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH), an agent that inhibits CYP-epoxygenase activity. The notion that the inhibitory effect of AA is mediated by CYP-epoxygenase–dependent metabolites is also supported by the observation that application of 200 nM 11,12-epoxyeicosatrienoic acid (EET) inhibited ENaC in the CCD. In contrast, addition of 5,6-, 8,9-, or 14,15-EET failed to decrease ENaC activity. Also, application of 11,12-EET can still reduce ENaC activity in the presence of MS-PPOH, suggesting that 11,12-EET is a mediator for the AA-induced inhibition of ENaC. Furthermore, gas chromatography mass spectrometry analysis detected the presence of 11,12-EET in the CCD and CYP2C23 is expressed in the principal cells of the CCD. We conclude that AA inhibits ENaC activity in the CCD and that the effect of AA is mediated by a CYP-epoxygenase–dependent metabolite, 11,12-EET.

Arachidonic acid and anandamide have opposite modulatory actions at the glycine transporter, GLYT1a

The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo-oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1-methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212-2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.

K depletion enhances the extracellular Ca2+-induced inhibition of the apical K channels in the mTAL of rat kidney.

We have shown previously that raising extracellular Ca(2)+ inhibited the apical 70-pS K channel in the thick ascending limb (TAL; Wang, W.H., M. Lu, and S.C. Hebert. 1996. Am. J. Physiol. 270:C103-C111). We now used the patch-clamp technique to study the effect of increasing the extracellular Ca(2)+ on the 70-pS K channel in the mTAL from rats on a different K diet. Increasing the extracellular Ca(2)+ from 10 microM to 0.5, 1, and to 1.5 mM in the mTAL from rats on a K-deficient (KD) diet inhibited the channel activity by 30, 65, and 90%, respectively. In contrast, raising the extracellular Ca(2)+ to 1.5 mM had no significant effect on channel activity in the mTAL from animals on a high K (HK) diet and further increasing the extracellular Ca(2)+ to 2.5, 3.5, and 5.5 mM decreased the channel activity by 29, 55, and 90%, respectively. Inhibition of the cytochrome P450 monooxygenase completely abolished the effect of the extracellular Ca(2)+ on channel activity in the mTAL from rats on a different K diet. In contrast, blocking cyclooxygenase did not significantly alter the responsiveness of the 70-pS K channel to the extracellular Ca(2)+. Moreover, addition of sodium nitropruside, a nitric oxide (NO) donor, not only increased the channel activity, but also blunted the inhibitory effect of the extracellular Ca(2)+ on the 70-pS K channel and decreased 20-hydroxyeicosatetraenoic acid (20-HETE) concentration in the mTAL from rats on a KD diet. In contrast, inhibiting NOS with L-NAME enhanced the inhibitory effect of the extracellular Ca(2)+ on the channel activity and increased 20-HETE concentration in the mTAL from rats on a high K diet. Western blot has further shown that the expression of inducible NO synthase (iNOS) is significantly higher in the renal medulla from rats on an HK diet than that on a KD diet. Also, addition of S-nitroso-N-acetylpenicillamine abolished the inhibitory effect of arachidonic acid on channel activity in the mTAL, whereas it did not block the inhibitory effect of 20-HETE. We conclude that a low dietary K intake increases the sensitivity of the 70-pS K channel to the extracellular Ca(2)+, and that a decrease in NOS activity is involved in enhancing the inhibitory effect of the extracellular Ca(2)+ on channel activity in the mTAL during K depletion.

Inhibition of Adenylylcyclase Activity in Mouse Cerebellum Membranes upon Hydrolysis of Triacylglycerols by Triacylglycerol Lipase, but Not Phospholipids by Phospholipase A2

We previously showed that arachidonic acid and related unsaturated free fatty acids (U-FFAs) inhibit the activity of adenylylcyclase in brain membranes of mice. The level of U-FFAs elevates when the hydrolysis of triacylglycerols (TAGs) and phospholipids is promoted. In this study, we examined whether activation of triacylglycerol lipase (TAG lipase) and phospholipase A2 (PLA2) results in the inhibition of adenylylcyclase activity in cerebellum membranes of mice. Incubation of Intralipos with TAG lipase in the presence of membranes mainly released oleic acid and linoleic acid and caused ≥95% inhibition of adenylylcyclase activity. In contrast, PLA2, though releasing substantial amounts of U-FFAs, increased the enzymatic activity. To account for this difference, we examined how by-products formed in U-FFA release by TAG lipase and PLA2 operated on the arachidonic acid-induced inhibition. Lysophosphatidylcholne and some other lysophospholipids, produced by PLA2, enhanced the adenylylcyclase activity and attenuated the inhibitory effect of arachidonic acid. On the other hand, no such effects were found with by-products of TAG lipase-mediated lipolysis. Rather, monoacylglycerols having U-FFAs, possibly formed by TAG lipase, potentiated the arachidonic acid-induced inhibition of adenylylcyclase. Bovine serum albumin, added into the mixture for the pretreatment of membranes with TAG lipase, prevented the inhibition of adenylylcyclase. These results indicate that by-products formed in U-FFA release have a crucial role for the U-FFA's action on adenylylcyclase and that U-FFAs released from TAG are an inhibitor of adenylylcyclase. It may be that albumin in plasma, and thus FFA-binding proteins within cells, are of importance in protecting adenylylcyclase upon U-FFA release.