Addition Of Nordihydroguaiaretic Acid Research Articles

Comparative spectroscopic analysis of nordihydroguaiaretic acid and related natural products to inhibition of calcium oxalate calculi

The prevalence of urolithiasis is still increasing worldwide. The goal of this study is to achieve a better understanding, from a comparative spectroscopic perspective, of the differences provided by chemical and natural products to the inhibitory processes of calculi formation. The research presented here places primary emphasis on the assessment of nordihydroguaiaretic acid (NDGA), the main chemical extract of the plant Larrea Tridentata, as a preventative to calcium oxalate crystal formation. It is also a logical continuation of our previous efforts using a traditional medicine approach to study such prevention. Complementary Raman and Fourier transformed infrared (FTIR) absorption analysis were used to provide a complete overview of morphological changes of calcium oxalate crystals, which were grown without and with the addition of NDGA at different concentrations. A gel diffusion technique was employed for sample preparation. Our results from both Raman and FTIR spectroscopies demonstrate that no structural transformation from a monohydrate to dehydrate occurs upon crystallization with NDGA, as we previously reported for the use of the natural plant infusion. Calculi synthesized with NDGA shows a distorted monohydrate morphology. Different effects inhibitory to calcium oxalate calculi formation are observed and analyzed here using chemical and natural extracts. If magnesium is the key to this difference, this study confirms its importance, not only in hundreds of metabolic pathways, but also in nephrological ones.

Nordihydroguaiaretic acid inhibition of NFATc1 suppresses osteoclastogenesis and arthritis bone destruction in rats

Nordihydroguaiaretic acid (NDGA) is known to have prominent anticancer activity against several cancers, and is also known to be an inhibitor of 5-lipoxygenase (5-LO). In this study, we investigated the regulatory function of NDGA on inflammatory bone destruction mediated by osteoclasts. NDGA markedly inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced formation of osteoclasts in cultures of murine osteoclast precursor cell line RAW-D cells and primary bone marrow-derived macrophages culture systems. The inhibitory effect of NDGA on osteoclastogenesis did not arise from the inhibition of 5-LO activity. NDGA did not affect MAPKs, such as p38, JNK, and NF-κB, but significantly inhibited the induction of NFATc1, a key transcription factor for osteoclastogenesis. NDGA also suppressed activation of ERK in osteoclast precursors. RANKL-induced calcium oscillation observed in osteoclast precursors was completely diminished by the addition of NDGA. In mature osteoclasts, RANKL-induced nuclear translocation of NFATc1 was clearly inhibited by NDGA treatment. Finally, in vivo studies demonstrated that administration of NDGA significantly reduced severe bone destruction and osteoclast recruitment in the ankle joint of rats with adjuvant-induced arthritis. These results indicate the potential utility of NDGA as a therapeutic agent for ameliorating inflammatory bone destruction in rheumatoid arthritis.

Effects of nordihydroguaiaretic acid on in vitro fermentation profiles of rumen bacteria1

Nordihydroguaiaretic acid (NDGA) is a secondary plant metabolite with antimicrobial properties, and therefore may have potential as a rumen modifier. Two in vitro experiments were conducted to determine the usefulness of NDGA as a rumen modifier. Exp. 1 evaluated the effect of adding 0, 5, 10, 50, and 100 mg/mL NDGA on growth of pure and mixed cultures of rumen bacteria. Growth of all cultures except Butyrivibrio fibrisolvens H17c was inhibited at 50 mg/mL NDGA (P < 0.05). Cultures from whole rumen fluid and B. fibrisolvens H17c were inhibited with 100 mg/mL NDGA (P < 0.05). Exp. 2 evaluated additions of NDGA on IVDMD (48 h) and VFA production. Three dietary substrates simulating different resources available for livestock production and 5 concentrations of NDGA were compared with monensin (47.5 μg/mL, MON, Elanco Animal Health, Indianapolis, IN). Substrates included (DM basis) 100% meadow hay (H100), 50% alfalfa with 50% ground corn (H50), and 90% ground corn with 10% alfalfa (H10). Treatments were 0 (Control; CON), 20, 40, 60, 80 μg/mL NDGA and MON. Treatment means were compared using 2 single degree of freedom contrasts (0 μg/mL NDGA vs. MON and NDGA vs. MON) and orthogonal polynomial contrasts within NDGA concentrations. Monensin fermented with H100 had the least (P < 0.01) IVDMD. A linear increase in IVDMD was observed for H50 (P < 0.01) but not H10 or 100 (P > 0.40). Acetate was quadratic for all substrates tested with NDGA (P < 0.01) and adding NDGA vs. MON resulted in 9% greater values (P < 0.01). Propionate increased by addition of MON compared with CON, which was opposite for acetate. Propionate showed the greatest increase with addition of MON and was dependent on diet vs. CON (H100 vs.H50 vs. H10; 22.5%, 44.4%, and 30.2%, respectively). When H100 was used, total VFA declined linearly by 61% with increasing NDGA (P < 0.01), whereas H50 and H10 were quadratic (P < 0.01) with the greatest total VFA resulting from 40 and 80 mg/mL NDGA for H50 and H10, respectively. Addition of NDGA tended to decrease total VFA (P =0.06) for H100 and H10 by 18.5% and 9.0%, respectively; however, H50 did not differ (P = 0.82) compared with MON. Butyrate increased linearly with increasing NDGA for H10 (P < 0.03) and quadratic for H50 and H100 (P < 0.01). The lowest overall acetate:propionate ratio was obtained with addition of MON to H10 (1.35) and the greatest ratio resulted from adding 60 μg/mL NDGA to H100 (3.63). Rumen fermentation was responsive to NDGA, and the response is dependent on diet.

Inhibition of L-type Ca2+ channel current and negative inotropy induced by arachidonic acid in adult rat ventricular myocytes

We have previously shown an increase in arachidonic acid (AA) release in response to proinflammatory cytokines in adult rat ventricular myocytes (ARVM). AA is known to alter channel activities; however, its effects on cardiac L-type Ca(2+) channel current (I(Ca,L)) and excitation-contraction coupling remain unclear. The present study examined effects of AA on I(Ca,L), using the whole cell patch-clamp technique, and on cell shortening (CS) and the Ca(2+) transient of ARVM. I(Ca,L) was monitored in myocytes held at -70 mV and internally equilibrated and externally perfused with Na(+)- and K(+)-free solutions. Exposure to AA caused a voltage-dependent block of I(Ca,L) concentration dependently (IC(50) 8.5 microM). The AA-induced inhibition of I(Ca,L) is consistent with its hyperpolarizing shift in the voltage-dependent properties and reduction in maximum slope conductance. In the presence of AA, BSA completely blocked the AA-induced suppression of I(Ca,L) and CS. Intracellular load with AA had no effect on the current density but caused a small depolarizing shift in the I(Ca,L) activation curve, suggesting a site-specific action of AA. Moreover, intracellular AA had no effect on the extracellular AA-induced decrease in I(Ca,L). Pretreatment with indomethacin, an inhibitor of cyclooxygenase, or addition of nordihydroguaiaretic acid, an inhibitor of lipoxygenase, had no effect on AA-induced changes in I(Ca,L). Furthermore, AA suppressed CS and Ca(2+) transients of intact ARVM with no significant effect on SR function and myofilament Ca(2+) sensitivity. Therefore, these results suggest that AA inhibits contractile function of ARVM, primarily due to its direct inhibition of I(Ca,L) at an extracellular site.

Nordihydroguaiaretic acid (NDGA) blocks the differentiation of C2C12 myoblast cells

Addition of nordihydroguaiaretic acid (NDGA) to the differentiation medium of C2C12 mouse myoblast cells caused severe inhibition of the formation of myotubes and suppressed differentiation-dependent elevation in the levels of the creatine kinase M isozyme (CKM). Under these conditions, NDGA did not cause significant increase of damaged cells, as detected by annexin-V-FITC assay, or induction of heat shock proteins, known to be a response against extracellular stress. The results suggest that NDGA itself is not toxic but can effectively blocks the differentiation-dependent increase of CKM during C2C12 differentiation. The levels of muscle specific bHLH proteins MyoD, Myf5, and myogenin were also decreased by addition of NDGA, indicating a block of the initial step of the myogenesis through downregulation of muscle specific genes. NDGA is known to be a lipoxygenase inhibitor but other examples, like MK-886 and CDC, did not exert the same effects on differentiation of muscle cells, indicating that mechanisms of NDGA action are independent of its influence on lipoxygenase.

Lipoxygenase inhibitor-induced apoptosis in Madin–Darby canine kidney cells

Both cyclooxygenase (COX) and lipoxygenase (LOX), as well as their metabolites, are suggested to play a dual role in regulating cell survival and apoptosis in various types of cells. However, their mechanism remains unclear. When Madin–Darby canine kidney (MDCK) cells were treated with 12- O-tetradecanoylphorbol β-acetate (TPA) and nordihydroguaiaretic acid (NDGA), a LOX inhibitor, we detected a synergistic stimulation of apoptosis as determined by hallmarks such as DNA ladder formation and chromatin condensation. The apoptosis was slightly induced by TPA alone. The effect of TPA was markedly stimulated along with NDGA. NDGA on its own did not have the ability to induce apoptosis. Aspirin, a COX inhibitor, was not effective. Alternatively, hydrogen peroxide-induced apoptosis was not promoted by the addition of NDGA. These two types of apoptosis would be regulated by a different mechanism. Although NDGA-induced apoptosis might be associated with the depletion of intracellular reduced glutathione in some kinds of tumor cells, N-acetyl cysteine (NAC), a thiol agent, had no inhibitory effect on the NDGA-induced apoptosis. To determine the further mechanism of NDGA-induced apoptosis in MDCK cells, we are examining the involvement of apoptosis-specific signaling molecules and the effect of lipoxygenase metabolites on NDGA-induced apoptosis.

Glutathione oxidation and mitochondrial depolarization as mechanisms of nordihydroguaiaretic acid-induced apoptosis in lipoxygenase-deficient FL5.12 cells.

Nordihydroguaiaretic acid (NDGA) induces apoptosis in a variety of cell lines. The mechanism(s) of this effect is not known, although the focus has been on the ability of NDGA to inhibit lipoxygenase (LOX) activities. In the present study, NDGA-induced apoptosis was studied in a murine hematopoietic cell line, FL5.12. Although this cell line lacks detectable LOX protein or activities, NDGA (10 microM) was able to induce apoptosis. There was a massive loss of mitochondrial membrane potential by 4 h after the addition of NDGA, suggesting that this organelle might be targeted by NDGA. A pro-oxidant NDGA effect has been suggested as playing a role in apoptosis. This was supported by the findings that glutathione disulfide levels were increased by 4 h following treatment with 10 microM NDGA, that pretreatment with N-acetylcysteine completely blocked the NDGA-induced loss of membrane potential and apoptosis, and that lipid peroxidation was enhanced in cells treated with NDGA. However, no evidence of increased levels of reactive oxygen could be seen in NDGA-treated cells loaded with dichlorofluorescin diacetate or dihydrorhodamine and analyzed by flow cytometry. Bcl-X(L) protein levels were unaffected by NDGA treatment. Caspase-3 was rapidly activated with a peak at 8 h after FL5.12 cells were treated with NDGA. Ac-DEVD-CHO (25 microM) and boc-asp-FMK (20 microM) both inhibited caspase-3 enzyme activity by 97% 8 h after NDGA treatment. Boc-asp-FMK, a more general caspase inhibitor, delayed NDGA-induced apoptosis while Ac-DEVD-CHO, a more specific inhibitor of caspase-3, had no effect. These results suggest that NDGA-induced apoptosis happens through reactions that depolarize mitochondria, oxidize glutathione and lipids, but do not generate significant amounts of free reactive oxygen species.

Expression of 15-Lipoxygenase by Human Colorectal Carcinoma Caco-2 Cells during Apoptosis and Cell Differentiation

We studied arachidonic acid metabolism and the expression of cyclooxygenase (Cox) and 15-lipoxygenase (15-LO) in the human colorectal carcinoma cell line, Caco-2, which undergo apoptosis and cell differentiation in the presence of sodium butyrate (NaBT). Caco-2 cells expressed very low levels of Cox-1 but highly expressed Cox-2. NaBT treatment shifted the arachidonic acid metabolites by cell lysates from prostaglandins to 15-hydroxyeicosatetraenoic acid, indicating the presence of a 15-LO. Linoleic acid, an excellent substrate for 15-LO, was metabolized poorly by the Caco-2 cells, but NaBT treatment shifted metabolism to 15-LO metabolite, 13(S)-hydroxyoctadecadienoic acid. Caco-2 cells expressed a 15-LO but only after treatment with NaBT, as determined by Northern blotting. Immunoblotting with anti-human 15-LO antibody detected a 72-kDa band in NaBT-treated Caco-2 cells. Expression of 15-LO mRNA was dependent on the duration of NaBT treatment, with the highest expression observed between 10 and 24 h. Results from expression and metabolism studies with arachidonic and linoleic acid cells indicated Cox-2 was responsible for the lipid metabolism in control cells, whereas 15-LO was the major enzyme responsible after NaBT induction of apoptosis and cell differentiation. The 15-LO in Caco-2 cells was characterized as human reticulocyte 15-LO by reverse transcription-polymerase chain reaction and restriction enzyme analysis. The expression of 15-LO and 15-hydroxyeicosatetraenoic acid or 13(S)-hydroxyoctadecadienoic acid formation correlates with cell differentiation or apoptosis in Caco-2 cells induced by NaBT. The addition of nordihydroguaiaretic acid, a lipoxygenase inhibitor, significantly increased NaBT-induced apoptosis, whereas the addition of indomethacin did not alter NaBT-induced apoptosis in the Caco-2 cells. However, indomethacin treatment decreased the expression of Cox-2 in NaBT-treated cells and significantly increased the expression of 15-LO during NaBT treatment. These studies suggest a role for 15-LO, in addition to Cox-2, in modulating NaBT-induced apoptosis and cell differentiation in human colorectal carcinoma cells.

Arachidonic acid increases cholinergic secretory responsiveness of ferret tracheal glands.

The purpose of this study was to determine if arachidonic acid could alter ferret tracheal gland secretory responsiveness to a cholinergic agonist. We prepared glandular explants and incubated the explants in medium containing [3H]glucosamine. Secretory responsiveness was expressed as the percent change in basal secretion of acid-precipitable [3H]glucosamine-labeled glycoconjugates induced by the addition of agonist with and without arachidonic acid [mean +/- SE (n)]. Addition of 10(-3) M arachidonic acid caused a significant increase in secretion [28 +/- 6% (n = 6)] compared with untreated control tissues [-10 +/- 4% (n = 7), P less than or equal to 0.05]. Carbachol (10(-7) M) increased secretion 39 +/- 9% (n = 7). The combination of 10(-3) M arachidonic acid and 10(-7) M carbachol elicited a significantly greater change in secretion compared with either agent alone [173 +/- 50% (n = 5)]. The addition of nordihydroguaiaretic acid (10(-6) M) or indomethacin (10(-6) M) partially attenuated the arachidonic acid-enhanced secretory responsiveness to carbachol. Treatment with both blockers completely inhibited the arachidonic acid-enhanced secretory responsiveness to carbachol. The effect of arachidonic acid on cholinergic stimulation was also abolished by treating the explant cultures with tetrodotoxin (10(-7) M). This hypersecretory state is most likely mediated by eicosanoid-induced release of neurotransmitters from nerve terminals.

Antimutagenic and antitumorigenic activities of nordihydroguaiaretic acid

Nordihydroguaiaretic acid (NDGA), which occurs in the resinous exudates of many plants is used as an antioxidant in fats and oils. In this study we show that NDGA inhibited the mutagenicity of methyl methanesulfonate, benzo[ a]pyrene (BP), 2-aminofluorene, and aflatoxin BP 1 in Salmonella typhimurium strain TA100 or TA98 in the absence and presence of rat hepatic microsomal activation system. The addition of NDGA during and after nitrosation of methylurea (MU) resulted in a dose-dependent inhibition of mutagenicity induced by nitrosation products of MU. In a two-stage skin tumorigenesis protocol using 7,12-dimethylbenz[ a]anthracene (DMBA) as the initiating agent followed by twice weekly applications of 12- O-tetradecanoylphorbol-13-acetate (TPA) as tumor promoter, pretreatment of animals with NDGA prior to DMBA application, afforded significant protection against skin tumorigenicity in female SENCAR mice. In additional studies, skin application of NDGA also inhibited the binding of topically applied 3HBP and 3HDMBA to epidermal DNA. When assessed in the anti-tumor promotion protocol, pretreatment of animals with NDGA before each application of TPA in DMBA-initiated mouse skin, resulted in 72% decrease in the total number of tumors when compared to non-NDGA pretreated animals. The possible mechanism(s) of the antimutagenic and anti-tumorigenic activities may be due to the multiple effects of NDGA as inhibitor of the carcinogen metabolism and DNA-adduct formation, scavenger of carcinogen free radicals, and as inhibitor of TPA-induced ornithine decarboxylase activity.

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca 2+]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca 2+]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca 2+ probe. Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca 2+]i in both small and large luteal cells, having an effect at concentrations as low as 5μM; and was maximally effective at 50μM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca 2+]i, with NDGA exerting a greater stimulation of [Ca 2+i than indomethacin. Prostaglandin F 2α (PGF 2α) as well as prostaglandin E 2 (PGE 2) increased [Ca 2+ in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca 2+]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF 2α and PGE 2) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca 2+]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.

Role of lipoxygenase in the mechanism of acrosome reaction in mammalian spermatozoa

The acrosome reaction (AR) in bull spermatozoa was induced by the Ca 2+-ionophore A23187, by dilauroylphosphatidylcholine or by arachidonic acid in the presence of Ca 2+ in the incubation medium. The occurrence of AR was determined by following the release of acrosin from the cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of both lipoxygenase and prostaglandin-synthetase, caused 35%, 43% and 69% inhibition of AR at concentrations of 1, 10 or 100 μM, respectively. Eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, caused 17%, 61% and 77% inhibition of AR at concentrations of 20, 40 or 80 μg/ml, respectively. When AR was induced by arachidonic acid, ETYA, causes 36% and 58% inhibition at concentrations of 2 or 20 μg/ml, respectively. Under identical conditions, 100 μM indomethacin, a specific inhibitor of prostaglandin-synthetase, showed no inhibition but rather 35% stimulation at acrosin release rate. The fact that AR is inhibited by NDGA and not by indomethacin indicates that the lipoxygenase, rather than prostaglandin-synthetase, is involved in the mechanism of AR. Since the inhibition by NDGA is seen in the presence of the Ca-ionophore, we suggest that lipoxygenase activity is not involved in enhancing calcium transport into the cell, but rather at other steps in AR mechanism. A thin-layer chromatography revealed the presence of 15-HETE, the classical product of 15-lipoxygenase activity, which was identified by HPLC. Under AR conditions, there is an elevation of lipoxygenase products and the addition of NDGA caused a reduction in their levels. The inhibition of acrosin release by NDGA can be eliminated by adding 15-HETE or 15-HPETE to the incubation medium. In conclusion, we suggest here for the first time, a physiological role for 15-lipoxygenase in the mechanism of AR in mammalian spermatozoa.

Cyclooxygenase Inhibition Potentiates the Pulmonary Vascular Responses to Ethanol in Dogs

The effects of ethanol on pulmonary hemodynamics and the role of arachidonic acid metabolites in mediating these responses were studied in the isolated, blood-perfused canine lung lobe. Ethanol added directly to the venous reservoir at a concentration of 0.01 or 0.1% did not alter the pulmonary vascular resistance, however, 1.0% ethanol increased (p less than 0.05) the pulmonary resistance. Indometacin, a cyclooxygenase inhibitor, significantly increased baseline pulmonary vascular resistance and markedly potentiated the pulmonary vascular responses to the three concentrations of ethanol. Addition of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, followed by indometacin, did not diminish the indometacin-induced increases in basal tone or enhancement of the responses to ethanol indicating that leukotrienes are not involved in mediating the responses to ethanol. These results suggest that in isolated, blood-perfused dog lung lobe, a high concentration of ethanol increases the pulmonary vascular resistance and that cyclooxygenase inhibition markedly potentiated the responses to ethanol by reducing the production of vasodilator prostaglandin, most probably prostacyclin.

Effect of dietary restriction on triglyceride levels in the uterus isolated from pregnant rats. Influences of prostaglandins and indomethacin

Triglyceride (TGs) concentrations in uterine strips isolated from 14 or from 21 days-pregnant rats, either normal-fed or following a restricted-diet rats (50% food intake for 14 days), were measured. Determinations were made immediately after killing (0 min time or post-isolation) as well as after a period of incubation in glucose-free medium (60 min time or post-incubation). The post-isolation levels of TGs (0 min) in the uterus from normal-fed animals at 14 or at 21 days of pregnancy, were significantly higher in implantation sites than in the interembryonic segments. These values of TGs (0 time) did not change, in comparison to post-incubation concentrations (60 min), either without additions or in the presence of indomethacin (5 × 10 −6 M) or of prostaglandins (PGs) E 1, E 2 or F 2α (10 −7 M). At 0 time, uterine TGs of rats subjected to dietary restriction, increased as pregnancy progressed, more than in normal-fed controls. The post-incubation (60 min) pattern was different depending on the days of pregnancy; i.e. at 14 days, incubation in Krebs-Ringer Bicarbonate-medium (KRB) led to a significant fall unaffected by the addition of propranolol (10 −6 M). However, in the presence of indomethacin, TGs values had a level similar to the initial one (0 time). Furthermore, exogenous PGE 1 or PGE 2 failed to alter the effect of indomethacin, as PGF 2α did. In the group of pregnant rats at term, fed a restricted-diet for 14 days prior to delivery, the initial values remained unaltered at the end of the incubation period without additions, and did not decline in the presence of indomethacin, either alone or plus PGE 1, PGE 2 or PGF 2α. The addition of nordihydroguaiaretic acid did not alter TGs levels, and the same was the case when alpha-adrenergic blockers, namely phentolamine (10 −6 M) or yohimbine (5 × 10 −6 M) were added. At 6 days post-partum, isolated uteri from normal-fed rats exhibited TGs levels similar to controls (0 time), after 60 min of incubation in glucose-free KRB-medium, with or without indomethacin addition. On the contrary, 6 days after parturition, the levels of TGs in uteri from restricted-diet rats declined significantly after 60 min of incubation. The results suggest an interesting relationship between dietary restriction in pregnancy and uterine lipid metabolism, as reflected by uterine tissue TGs levels.

Differential effects of anticholinergic drugs on platelet aggregation induced by platelet-activating factor and other agents

formation of various lipoxygenase products. A typical radiochromatographic scan obtained after t.1.c. of the products of incubation is shown in Fig. 1 . The lipoxygenase products were identified by co-migration with authentic hydroxy acid standards. A characteristic pattern of one major ( K , 0.6) and one minor ( K , 0.15) peak can be seen (Fig. 1). The major compound co-migrated with 1 2-hydroxyeicosatetraenoic acid. When 5-hydroxytryptamine (1-100 p ~ ) was added to the incubation mixture, mean 12-hydroxyeicosatetraenoic acid production exceeded the control value by 1.15-fold to 4.1-fold in a concentration-related manner ( I ’ < 0.001. t i = 7). Addition of nordihydroguaiaretic acid, an inhibitor of lipoxygenase activity, and ketotifen to the incubation mixture caused a concentration-dependent inhibition of the conversion of arachidonic acid to lipoxygenase products by human platelets. The mean values ( ~ s . E . M . ) for inhibiting the production of 12-hydroxyeicosatetraenoic acid by SOo% ( lC5,,) for ketotifen and nordihydroguaiaretic acid were 79 k 9 and 35 k 4 p ~ , respectively. lndomethacin (up t o 2 0 0 PM), an inhibitor of fatty acid cyclo-oxygenase activity, had no inhibitory effect on 5-hydroxytryptamine-induced stimulation o f 12-hydroxyeicosatetraenoic acid production, whereas it stimulated the production o f trihydroxyeicosatrienoic acid ( K , 0. 15) 3-fold. In separate experiments we have found that ketotifen also inhibits rat lung arachidonate lipoxygenase and soybean lipoxygenase activities. These experiments demonstrate that ketotifen inhibits arachidonate lipoxygenase activity in platelets. Since 12hydroxyeicosatetraenoic acid and its labile precursor 12hydroperoxyeicosatetraenoic acid are potent chemotactic and vasoactive agents (Goetzl & Sun, 1Y79), we suggest that ketotifen may directly influence S-hydroxytryptamineinduced stirnulation of platelet arachidonate lipoxygenase activity.

Role of arachidonic acid in luteinizing hormone-releasing hormone action: stimulation of progesterone production in rat granulosa cells.

This study investigates the hypothesis that stimulation of ovarian progesterone (P) production by LHRH is mediated in part by arachidonic acid (AA). In rat granulosa cells prelabeled with [3H]AA or [3H]inositol, treatment with LHRH stimulates the accumulation of radiolabeled inositol phosphosphates, diacyglycerol, and unesterified AA. Treatment with AA (3 X 10(-7)-10(-5) M) enhances P production in a dose-dependent manner. Concurrent treatment with AA and LHRH (or a LHRH agonist) further stimulates P production. The stimulatory effect of AA on P production, either alone or in combination with LHRH, could be seen as early as 3 h after AA addition. Addition of a phorbol ester, 12-O-tetracecanoylphorbol-13-acetate (TPA), to granulosa cells stimulates P production. Interestingly, the concomitant presence of AA and TPA further enhances production compared with either TPA or AA treatment alone. The stimulatory effect of AA on P production is completely abolished by nordihydroguaiaretic acid, but not by indomethacin. On the other hand, addition of nordihydroguaiaretic acid (but not indomethacin) reduces LHRH-stimulated P levels by about 50%. Nordihydroguaiaretic acid, as well, markedly attenuates the P response due to combined treatment with LHRH and AA. Taken together, these results strongly support the notion that AA (or its lipoxygenated metabolites) partially mediates the action of LHRH. Along with the calcium and protein kinase-C pathways, AA metabolism may be involved in the biological actions of LHRH in the ovary.