Stimulatory Effect Of Arachidonic Acid Research Articles

Adipokines secretion in feline primary adipose tissue culture in response to dietary fatty acids

BackgroundObesity in cats has been associated with alterations in adipokines including: adiponectin, interleukin-6 (IL6), and tumor necrosis factor-α (TNFα). Omega-3 polyunsaturated fatty acids have multiple beneficial effects on obesity-associated disorders, and therefore may alleviate these alterations. This study aimed to determine the effects of body condition, fat depot, troglitazone, and different fatty acids on secretion of adiponectin, IL6 and TNFα from adipose tissue of healthy cats. Subcutaneous and visceral adipose tissue samples were collected from 18 healthy intact female cats, and body condition score (Range 3–7/9) was determined. Concentrations of adiponectin were measured in mature adipocytes cultures and concentrations of IL6 and TNFα were measured in stromovascular cells cultures following treatment with control medium, troglitazone at 10 μM, eicosapentaenoic acid, arachidonic acid, or palmitic acid, at 25, 50, or 100 μM.ResultsStromovascular cells of visceral origin secreted higher concentrations of IL6 than corresponding cells of subcutaneous origin (P = 0.003). Arachidonic acid treatment at 25, 50, and 100 μM increased IL6 secretion in subcutaneous (P = 0.045, P = 0.002, and P < 0.001, respectively) and visceral (P = 0.034, P = 0.001, and P < 0.001, respectively) stromovascular cells. Eicosapentaenoic acid treatment increased TNFα secretion in subcutaneous stromovascular cells at 25, 50, and 100 μM (P = 0.002, P = 0.001, and P = 0.015, respectively) and in visceral stromovascular cells at 50 μM (P < 0.001). No significant effect on medium adiponectin concentration was observed following troglitazone treatment (P = 0.4) or fatty acids treatments at 25 (P = 0.2), 50 (P = 0.8), or 100 (P = 0.7) μM. Body condition score did not have significant effects on medium concentrations of adiponectin (P = 0.4), IL6 (P = 0.1), or TNFα (P = 0.8).ConclusionsThis study demonstrated higher basal secretion of IL6 from visceral compared to subcutaneous adipose tissue, a stimulatory effect of arachidonic acid on secretion of IL6 and a stimulatory effect of eicosapentaenoic acid on TNFα from feline adipose tissue.

AKT and cytosolic phospholipase A2α form a positive loop in prostate cancer cells.

Aberrant increase in protein kinase B (AKT) phosphorylation (pAKT), due to a gain-of-function mutation of phosphatidylinositol-3-kinase (PI3K) or loss-of-function mutation or deletion of phosphatase and tensin homolog (PTEN), is a common alteration in prostate cancer and associated with poor prognosis. Cytosolic phospholipase A2α (cPLA2α) is a lipid modifying enzyme by catalyzing the hydrolysis of arachidonic acid from membrane phospholipid. The released arachidonic acid and its metabolites contribute to survival and proliferation of prostate cancer cells. In this mini-review, we summarize the relationship between pAKT and cPLA2α in prostate cancer cells. There was a concordant increase in pAKT and cPLA2α levels in prostate tissue of prostate epithelial-specific PTEN-knockout mice compared to PTEN-wild type mice. Restoration of PTEN expression or inhibition of PI3K action decreased cPLA2α expression in PTEN-mutated or deleted prostate cancer cells. An increase in AKT phosphorylation elevated, whereas inhibition of AKT phosphorylation diminished, cPLA2α protein levels. pAKT had no influence on cPLA2α expression at mRNA levels but stabilized cPLA2α at protein levels by protecting it from degradation. Conversely, an induction of cPLA2α expression led to an increase in pAKT levels in PTEN-mutated or deleted prostate cancer cells, while silencing of cPLA2α expression or pharmacological blocking cPLA2α action decreased pAKT levels. The diminishment of pAKT by either genetic silencing or pharmacological blocking of cPLA2α was mitigated by the addition of arachidonic acid. The stimulatory effect of arachidonic acid on pAKT levels was lessened by inhibiting the production of arachidonic acid metabolites. These studies have revealed a link between oncogenic pathway and lipid metabolism and provided potential molecular targets for treating prostate cancer.

Altering n-3 to n-6 polyunsaturated fatty acid ratios affects prostaglandin production by ovine uterine endometrium

Consumption of n-3 polyunsaturated fatty acids (PUFAs) is considered beneficial to health but effects on fertility remain uncertain. This study investigated the effect of n-3 PUFA supplementation on endometrial prostaglandin (PG) production. Ovine uterine endometrial cells were cultured to confluence in DMEM/F12 medium containing 10% foetal bovine serum. Stromal and epithelial cell populations were confirmed by immunocytochemistry. Cultures were supplemented with 0, 20 or 100μM of α-linolenic acid (ALA), stearidonic acid (SDA), eicosapentaenoic acid (EPA) with lipopolysaccharide (LPS) at 0 and 0.1μg/ml, or different combinations of EPA with arachidonic acid (AA) in serum-free medium for 24h. PGs were quantified using radioimmunoassay and PG-endoperoxide synthase (PTGS) isoforms, PGE and PGF synthase (microsomal PGES1 and PGFS) mRNAs by qPCR. LPS increased PGE2 production significantly without changing PGF2α production, causing increased PGE2:PGF2α ratios. ALA and SDA increased PGE2, PGF2α and PGE2:PGF2α ratios (P<0.05–0.01) while EPA alone did not affect PG generation. AA significantly stimulated PTGS1 and PTGS2 mRNA expression and PGE2 and PGF2α production (P<0.01). The stimulatory effect of AA was attenuated by up to 80% (P<0.05) when AA was combined with EPA. The PGE2:PGF2α ratio was not affected by AA or EPA alone, but increased when these two PUFAs were combined (P<0.05). SDA and EPA decreased PTGS1 mRNA expression (P<0.05) but did not alter PTGS2 expression. EPA and AA up-regulated mPGES1 expression (P<0.05) without affecting PGFS expression. Since AA is preferentially incorporated in uterine endometrium to produce 2-series PGs, alteration of PG production by EPA may affect many reproductive processes.

Cytosolic phospholipase A2α sustains pAKT, pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A2α (cPLA2α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA2α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA2α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA2α decreased pERK and AR protein levels. The inhibitory effect of cPLA2α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA2α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA2α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.

Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain

We have been searching for a mechanism to induce smooth muscle contraction that is not associated with phosphorylation of the regulatory light chain (RLC) of smooth muscle myosin (Nakamura A, Xie C, Zhang Y, Gao Y, Wang HH, Ye LH, Kishi H, Okagaki T, Yoshiyama S, Hayakawa K, Ishikawa R, Kohama K. Biochem Biophys Res Commun 369: 135-143, 2008). In this article, we report that arachidonic acid (AA) stimulates ATPase activity of unphosphorylated smooth muscle myosin with maximal stimulation (R(max)) of 6.84 +/- 0.51 relative to stimulation by the vehicle and with a half-maximal effective concentration (EC(50)) of 50.3 +/- 4.2 microM. In the presence of actin, R(max) was 1.72 +/- 0.08 and EC(50) was 26.3 +/- 2.3 microM. Our experiments with eicosanoids consisting of the AA cascade suggested that they neither stimulated nor inhibited the activity. Under conditions that did not allow RLC to be phosphorylated, AA stimulated contraction of smooth muscle tissue with an R(max) of 1.45 +/- 0.07 and an EC(50) of 27.0 +/- 4.4 microM. In addition to the ATPase activities of the myosin, AA stimulated those of heavy meromyosin, subfragment 1 (S1), S1 from which the RLC was removed, and a recombinant heavy chain consisting of the myosin head. The stimulatory effects of AA on these preparations were about twofold. The site of AA action was indicated to be the step-releasing inorganic phosphate (P(i)) from the reaction intermediate of the myosin-ADP-P(i) complex. The enhancement of P(i) release by AA was supported by computer simulation indicating that AA docked in the actin-binding cleft of the myosin motor domain. The stimulatory effect of AA was detectable with both unphosphorylated myosin and the myosin in which RLC was fully phosphorylated. The AA effect on both myosin forms was suggested to cause excess contraction such as vasospasm.

Epoxyeicosatrienoic Acid Activates BK Channels in the Cortical Collecting Duct

The cortical collecting duct (CCD), which is involved in renal potassium (K) excretion, expresses cytochrome P450 (CYP)-epoxygenase. Here, we examined the effect of high dietary K on renal expression of CYP2C23 and CYP2J2 in the rat, as well as the role of CYP-epoxygenase-dependent metabolism of arachidonic acid in the regulation of Ca(2+)-activated big-conductance K (BK) channels. By Western blot analysis, high dietary K stimulated the expression of CYP2C23 but not CYP2J2 and increased 11,12-epoxyeicosatrienoic acid (11,12-EET) levels in isolated rat CCD tubules. Application of arachidonic acid increased BK channel activity, and this occurred to a greater extent in rats on a high-K diet compared with a normal-K diet. This effect was unlikely due to arachidonic acid-induced changes in membrane fluidity, because 11,14,17-eicosatrienoic acid did not alter BK channel activity. Inhibiting CYP-epoxygenase but not cyclooxygenase- or CYP-omega-hydroxylase-dependent pathways completely abolished the stimulatory effect of arachidonic acid on BK channel activity. In addition, application of 11,12-EET mimicked the effect of arachidonic acid on BK channel activity, even in the presence of CYP-epoxygenase inhibition. This effect seemed specific to 11,12-EET, because both 8,9- and 14,15-EET failed to stimulate BK channels. Finally, inhibition of CYP-epoxygenase abolished iberiotoxin-sensitive and flow-stimulated but not basal net K secretion in isolated microperfused CCD. In conclusion, high dietary K stimulates the renal CYP-epoxygenase pathway, which plays an important role in activating BK channels and flow-stimulated K secretion in the CCD.

Arachidonic acid stimulates internalisation of leptin by human placental choriocarcinoma (BeWo) cells

Arachidonic acid at 100 nM stimulated internalisation of 125I-leptin in human placental choriocarcinoma (BeWo) cells by 3-fold compared with controls. In contrast, eicosapentaenoic acid at similar concentration decreased internalisation of leptin by 2-fold. Use of ibuprofen and indomethacin (inhibitors of prostaglandin synthesis) inhibited the stimulatory effect of arachidonic acid. Prostaglandin E 2, a cyclooxygenase metabolite of arachidonic acid, stimulated internalisation of leptin by these cells. All these data demonstrate that stimulation of leptin internalisation by arachidonic acid in placental trophoblasts may be mediated via prostaglandin E 2.

Stimulatory Effect of Arachidonic Acid on T-47DHuman Breast Cancer Cell Growth Is Associated WithEnhancement of Cyclin D1 mRNA Expression

Experimental and human studies have provided evidence that a high intake of nB6 polyunsaturated fatty acids stimulates mammary carcinogenesis. Arachidonic acid, an nB6 fatty acid consumed in the diet or derived from dietary linoleic acid, is thought to play a key role in enhancement of mammary tumor development. In this study, we investigated the effects of arachidonic acid on T-47D breast cancer cell growth, cell cycle progression, and the expression of cyclin D1 mRNA. Our data show that arachidonic acid stimulated the growth of T-47D cells with a twofold stimulation at 5 μg/ml. This effect was associated with an increase in the proportion of cells in the S phase of the cell cycle and preceded by stimulation of the expression of cyclin D1 mRNA, with maximal induction at 5 μg/ml. Cyclin D1 mRNA levels were increased within two hours of treatment and were maximal at five hours. These results suggest that arachidonic acid may exert a stimulatory effect on breast cancer cell growth and that this effect possibly involves the induction of cyclin D1 gene expression leading to cell cycle progression.

Modulation of the expression of early genes by polyunsaturated fatty acids

Expression of early genes is a characteristic immediate cellular response to mitogenic or inflammatory stimulation. Various second messenger systems have been found to transduce the signal from the plasma membrane to the nucleus. Recent observations indicate that in addition to well characterized second messenger systems, polyunsaturated fatty acids, especially the n-6 fatty acid arachidonic acid and its endogenously produced metabolites affect the expression of early genes in different cell types. At least in fibroblasts, the stimulatory effect of arachidonic acid can be antagonized by n-3 polyunsaturated fatty acids. Further identification of the mechanisms through which polyunsaturated fatty acids modulate early gene expression and regulate subsequent cellular responses, like cell growth, may help to define novel concepts in the management of cardiovascular diseases.

Arachidonic Acid Stimulates Prostate Cancer Cell Growth: Critical Role of 5-Lipoxygenase

Arachidonic acid (5,8,11,14-eicosatetraenoic acid), a member of the omega-6 poly-unsaturated fatty acids, was found to be an effective stimulator of human prostate cancer cell growthin vitroat micromolar concentrations. Selective blockade of the different metabolic pathways of arachidonic acid (e.g. ibuprofen for cyclooxygenase, SKF-525A for cytochrome P-450, baicalein and BHPP for 12-lipoxygenase, AA861 and MK886 for 5-lipoxygenase, etc.) revealed that the growth stimulatory effect of arachidonic acid is inhibited by the 5-lipoxygenase specific inhibitors, AA861 and MK886, but not by others. Addition of the eicosatetraenoid products of 5-lipoxygenase (5-HETEs) showed stimulation of prostate cancer cell growth similar to that of arachidonic acid, whereas the leukotrienes were ineffective. Moreover, the 5-series of eicosatetraenoids could reverse the growth inhibitory effect of MK886. Finally, prostate cancer cells fed with arachidonic acid showed a dramatic increase in the production of 5-HETEs which is effectively blocked by MK886. These experimental observations suggest that arachidonic acid needs to be metabolized through the 5-lipoxygenase pathway to produce 5-HETE series of eicosatetraenoids for its growth stimulatory effects on human prostate cancer cells.

Stimulatory effect of arachidonic acid on the release of GABA in matrix-enriched areas from the rat striatum

Arachidonic acid was shown to stimulate the release of preloaded [3H]GABA from microdiscs of tissue punched out in matrix-enriched areas of the rat striatum. This effect, which was calcium- and dose-dependent, persisted in the presence of inhibitors of arachidonic acid catabolism. Other fatty acids were less or not effective. Arachidonic acid also inhibited [3H]GABA uptake into purified striatal synaptosomes, however the arachidonic acid-evoked release of [3H]GABA persisted following inhibition of the GABA neuronal uptake process. The stimulatory effect of arachidonic acid on GABA release may largely result from the activation of a protein kinase C since the arachidonic acid response was reduced by several protein kinase C inhibitors. Arachidonic acid also dose-dependently stimulated the release of preloaded [3H]GABA from purified striatal synaptosomes. Similar results were obtained when synaptosomes were previously incubated with [3H]glutamine to study the release of endogenously synthesized [3H]GABA. Further indicating a direct action of the fatty acid on GABAergic neurons, the arachidonic acid-induced release of [3H]GABA from microdiscs was not modified in the presence of the D1 dopaminergic antagonist SCH23390 or of glutamatergic antagonists. Finally, the release of [3H]GABA evoked by the combined application of NMDA and carbachol (a treatment known to markedly stimulate arachidonic acid formation) was reduced by inhibitors of phospholipase A2 further indicating that endogenously formed arachidonic acid significantly facilitates the release of GABA in the striatum.

Arachidonic Acid, but Not Sodium Nitroprusside, Stimulates Presynaptic Protein Kinase C and Phosphorylation of GAP‐43 in Rat Hippocampal Slices and Synaptosomes

Activation of protein kinase C (PKC) and phosphorylation of its presynaptic substrate, the 43-kDa growth-associated protein GAP-43, may contribute to the maintenance of hippocampal long-term potentiation (LTP) by enhancing the probability of neurotransmitter release and/or modifying synaptic morphology. Induction of LTP in rat hippocampal slices by high-frequency stimulation of Schaffer collateral-CA1 synapses significantly increased the PKC-dependent phosphorylation of GAP-43, as assessed by quantitative immunoblotting with a monoclonal antibody that recognizes an epitope that is specifically phosphorylated by PKC. The stimulatory effect of high-frequency stimulation on levels of immunoreactive phosphorylated GAP-43 was not observed when 4-amino-5-phosphonovalerate (50 microM), an N-methyl-D-aspartate (NMDA) receptor antagonist, was bath-applied during the high-frequency stimulus. This observation supports the hypothesis that a retrograde messenger is produced postsynaptically following NMDA receptor activation and diffuses to the presynaptic terminal to activate PKC. Two retrograde messenger candidates--arachidonic acid and nitric oxide (sodium nitroprusside was used to generate nitric oxide)--were examined for their effects in hippocampal slices on PKC redistribution from cytosol to membrane as an indirect measure of enzyme activation and PKC-specific GAP-43 phosphorylation. Bath application of arachidonic acid, but not sodium nitroprusside, at concentrations that produce synaptic potentiation (100 microM and 1 mM, respectively) significantly increased translocation of PKC immunoreactivity from cytosol to membrane as well as levels of immunoreactive, phosphorylated GAP-43. The stimulatory effect of arachidonic acid on GAP-43 phosphorylation was also observed in hippocampal synaptosomes. These results indicate that arachidonic acid may contribute to LTP maintenance by activation of presynaptic PKC and phosphorylation of GAP-43 substrate. The data also suggest that nitric oxide does not activate this signal transduction system and, by inference, activates a distinct biochemical pathway.

Possible sites of dopaminergic inhibition of gonadotropin release from the pituitary of a teleost fish, tilapia

The present study is an attempt to find sites of dopaminergic inhibition along the transduction cascades culminating in gonadotropin (GtH) release in a teleost fish, tilapia. Experiments were carried out on perifused pituitary fragments and in primary culture of trypsinized pituitary cells. Salmon GnRH, chicken GnRH I and II stimulated GtH release in culture with estimated ED 50 values of 15.56 pM, 2.55 nM and 8.65 pM, respectively. Apomorphine (APO; 1 μM) totally abolished this stimulation. Dopamine (DA; 1 μM) reduced both basal and GnRHa-stimulated GtH release from perifused pituitary fragments but did not alter the formation of cAMP. In a similar perifusion experiment DA abolished GtH release in response to forskolin (10 μM) with no reduction in cAMP formation. This indicates that one site of the dopaminergic inhibition is distal to cAMP formation, an indication not compatible with the classic characteristic of DA D 2 type mode of action. The inhibition of GtH release in culture, caused by 1 μM APO, the specific DA D 2 agonists LY 171555 (LY) or bromocryptine (BRCR) could not be reversed by activating protein kinase C (PKC) by DiC8 or the phorbol ester TPA. This would indicate a site for DA action distal to PKC. However, the stimulatory effect of arachidonic acid (AA; 50 μM) in perifusion was not reduced by DA (1 μM) or by APO, LY or BRCR in culture, which suggests a site for DA action proximal to AA formation. APO, LY and BRCR reduced GtH release in response to the Ca 2+ ionophore A23187, however, their inhibitory effect was reversed by 10 μM ionomycin. The stimulatory effect of ionophore A23187 differed from that of ionomycin in that A23187 was able to stimulate GtH release only in the presence of Ca 2+ in the medium, whereas ionomycin stimulated the release also in the absence of the ion, and even in the presence of EGTA. It is assumed that ionomycin at 10 μM promotes the mobilization of Ca 2+ from intracellular stores. As neither ionomycin nor AA caused any leakage of lactic dehydrogenase from cultured pituitary cells, the GtH released in response to this agonist is specific and cannot be attributed to damage of the cells' membrane. It is proposed that, in addition to sites distal to cAMP formation and proximal to AA formation, DA may inhibit GtH release from the pituitary of tilapia at sites distal to Ca 2+ influx and at a step proximal to Ca 2+ mobilization from intracellular sources.

Arachidonic acid and eicosapentaenoic acid stimulate type II pneumocyte surfactant secretion.

Arachidonic acid and its leukotriene metabolites have been shown to stimulate surfactant secretion by alveolar type II cells. The present study was undertaken to determine the effects of various unsaturated fatty acids, including eicosapentaenoic acid, on surfactant secretion. Surfactant secretion was expressed as the percent of [3H]choline-derived phospholipids released into culture medium by type II pneumocytes of adult rats. Consistent with the earlier findings, arachidonic acid stimulated secretion in a concentration-dependent fashion (3.5-21 microM), doubling baseline secretion at 21 microM. Eicosapentaenoic acid was found to be equally effective as arachidonic acid in stimulating secretion. A comparison with palmitic, oleic and linoleic acids revealed that highly unsaturated fatty acids stimulated secretion to the greatest extent. This was supported by a positive correlation between degree of unsaturation (i.e., 0, 1, 2, 4 and 5 double bonds) and stimulation of surfactant secretion. In the present study, exogenous leukotriene E4 (10(-13)-10(-6) did not stimulate surfactant secretion. Neither nordihydroguairetic acid (0.1 microM) nor indomethacin (0.1 microM) affected arachidonic acid-stimulated secretion. The stimulatory effects of arachidonic acid and eicosapentaenoic acid on surfactant secretion were related to the highly unsaturated nature of the fatty acids and were not mediated by increased levels of cyclic adenosine monophosphate or calcium.

Involvement of phospholipase A2 and arachidonic acid in the depolarization-evoked accumulation of Ca2+ in hippocampal mossy fiber nerve endings.

Depolarization-evoked increases in intraterminal free Ca2+ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca2+ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca2+ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca2+ channel blockers. In addition, the K(+)-dependent effects on Ca2+ concentrations were attenuated in the presence of phospholipase A2 inhibitors, but were mimicked by the phospholipase A2 activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca2+ were reduced by voltage-sensitive Ca2+ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca2+. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A2 and the subsequent accumulation of unesterified arachidonic acid.

Activation of protein kinase C by arachidonic acid selectively enhances the phosphorylation of GAP-43 in nerve terminal membranes

Arachidonic acid (AA), a cis-unsaturated fatty acid that activates certain subspecies of protein kinase C (PKC), has been proposed to act as a retrograde messenger in modifying the efficacy of synapses during long-term potentiation (LTP). One prominent PKC substrate of the nerve terminal membrane, GAP-43 (F1, B-50, neuromodulin), shows an increase in phosphorylation that correlates with the persistence of LTP. The present study investigated whether AA might exert its effects on presynaptic endings by modulating the phosphorylation of GAP-43 and other membrane-bound proteins. Using synaptosomal membranes from the rat cerebrocortex, in which in vivo relationships between protein kinases and their native substrates are likely to be preserved, we found that in the absence of Ca2+, AA exerted a modest effect on the phosphorylation of GAP-43 and several other proteins; however, when AA was applied in conjunction with Ca2+, GAP-43 showed a particularly striking response: at Ca2+ levels likely to exist at the nerve terminal membrane during synaptic activity (10(-7) to 10(-5) M), AA (50 microM) increased the sensitivity of GAP-43 phosphorylation to Ca2+ by an order of magnitude, and increased its maximal level of phosphorylation by 50%. At resting Ca2+ levels, AA potentiated the stimulation in GAP-43 phosphorylation produced by 4 beta-phorbol 12,13-dibutyrate, a diacylglycerol (DAG) analog. The stimulatory effect of AA and its synergistic interaction with Ca2+ were found to be mediated by PKC, since they were blocked by a specific peptide inhibitor of PKC, [Ala25]PKC(19-31), but were unaffected by an inhibitor of protein phosphatase activity or by scavengers of free radicals. Since GAP-43 has been implicated in the development and plasticity of synaptic relationships, the synergistic effects of AA and the intracellular signals Ca2+ and DAG on the phosphorylation of GAP-43 may serve as an AND gate to modify presynaptic function and/or structure in response to coincident pre- and postsynaptic activity.

Arachidonic Acid and Prostaglandin E2 Stimulate Testosterone Production by Goldfish Testis in Vitro

Studies were conducted to determine the effects of arachidonic acid (AA) and its metabolites on testosterone (T) production by goldfish testis pieces. AA (25-400 μM) stimulated a dose-related increase in T production and this effect was potentiated by cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. T production in response to AA was elevated after 2 hr and continued to increase up to 24 hr. The stimulatory effect of AA was strongly inhibited by indomethacin and ibuprofen which block cyclooxygenase activity, whereas nordihydroguaiaretic acid, a lipoxygenase inhibitor, had no effect. Eicosatetraynoic acid, which inhibits both cyclooxygenase and lipoxygenase activity, also attenuated AA-stimulated T production. Together, these results suggest that AA effects on T production are mediated by metabolite(s) of the cyclooxygenase pathway. Several cyclooxygenase products stimulate T production; their relative potency was PGE2 = PGE1 ⪢ PGI2 > PGF2α while PGD2 had no effect. Lipoxygenase products of AA metabolism, including 5-, 11-, 12- and 15-hydroxyeicosatetraenoic acid had no effect on T production. AA, PGE2, and hCG increased cAMP production, suggesting that these compounds mediate T production through the actions of the same signaling molecule. In support of this contention, PGE2 had no additive effect on T production stimulated by a maximal dose of hCG. These data suggest that a cyclooxygenase metabolite of AA, likely PGE2, is involved in the control of steroidogenesis in the goldfish testis.

The human sperm acrosome reaction does not depend on arachidonic acid metabolism via the cyclooxygenase and lipoxygenase pathways.

The objective of this study was to determine whether the metabolism of arachidonic acid via the cyclooxygenase pathway, the lipoxygenase pathway, or both has a pivotal role in the human sperm acrosome reaction. To do so, the stimulatory effect of arachidonic acid and a number of its metabolites, as well as the inhibitory effect of cyclooxygenase and lipoxygenase inhibitors on the acrosome reaction, was evaluated. Arachidonic acid, prostaglandin E2, and prostacyclin (PGI2) induced the acrosome reaction when added to 3-hour preincubated (capacitated) spermatozoa. The arachidonic acid-induced acrosome reaction was dependent upon extracellular calcium. Leukotriene B4 and 15-HPETE only induced the acrosome reaction when present throughout the preincubation period, indicating that they may enhance the capacitation process rather than the acrosome reaction. Thromboxane did not affect the acrosome reaction under any of the conditions tested. Inhibitors of cyclooxygenase (indomethacin, phenylbutazone) and lipoxygenase (phenidone, nordihydroguiaretic acid) or FPL 55712 (a leukotriene antagonist) did not prevent the arachidonic acid-stimulated acrosome reaction. Furthermore, 5, 8, 11, 14-eicosatetraynoic acid (ETYA), the acetylenic analog of arachidonic acid that inhibits arachidonic acid metabolism, induced an acrosome reaction equivalent to that of arachidonic acid. These results strongly suggest that the acrosome reaction induced by exogenous arachidonic acid is not mediated via either the cyclooxygenase pathway or the lipoxygenase pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C.

The role of arachidonic acid in the regulation of steroidogenesis in rat Leydig cells was studied. A dose- and time-dependent biphasic effect on maximal and submaximal LH- and dibutyryl-cAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LH-stimulated cAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid potentiated LH- and dibutyryl-cAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-cAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenase and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC.

Dopamine Inhibits Inositol Phosphate Production, Arachidonic Acid Formation, and Corticosteroid Release by Frog Adrenal Gland through a Pertussis Toxin-Sensitive G-Protein*

We have previously shown that dopamine-evoked inhibition of corticosteroid production from adrenocortical cells is mediated through a decrease in prostaglandin biosynthesis. Since the catecholamine did not alter the stimulatory effect of arachidonic acid, it was proposed that dopamine may inhibit the formation of arachidonate from glycerophospholipids. To test this hypothesis, the effect of dopamine on phosphoinositol lipid metabolism was investigated in frog interrenal (adrenal) tissue. In [3H]myo-inositol-prelabeled frog interrenal slices, a short pulse of dopamine (50 microM) induced a biphasic effect on inositol phosphate production: a transient (1-min) increase, followed by a sustained inhibition. Concurrently, dopamine induced a transient reduction followed by a sustained increase in polyphosphoinositides. A 10-min pulse of the D2 dopamine receptor agonist apomorphine (50 microM) elicited a significant inhibition of basal levels of inositol phosphates (tris-, bis-, and mono-), and an increase in plasma membrane phosphoinositol lipid contents. The inhibitory effect of dopamine on inositol phosphate formation and corticosteroid release was abolished by a 24-h incubation of interrenal slices with pertussis toxin. In [3H]arachidonic acid-prelabeled interrenal slices, dopamine also decreased diacylglycerol (DG) and arachidonic acid (AA) concentrations. A delay of 1 min was observed between inhibition of DG and arachidonate, suggesting that AA is probably generated from DG. We conclude that in the adrenal cortex, activation of dopamine D2 receptors is coupled to a phosphoinositide-specific phospholipase-C mediated via a pertussis toxin-sensitive G-protein. Taken together, our data indicate that inhibition of inositol phosphate and AA formation is one of the mechanisms by which dopamine controls corticosteroid production by adrenocortical cells.