15-Lipoxygenase products stimulate prolactin secretion from a cloned strain of rat pituitary cells

Arachidonic acid and its lipoxygenase products may contribute to the process of prolactin (PRL) release. In the present study we investigate the role of 15-hydroperoxyeicosatetraenoic acid (15-HPETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on PRL secretion from GH3 cells. The incubation of GH3 cells with the lipoxygenase product 15-HETE significantly increased PRL release in a concentration-dependent manner. Nordihydroguaiaretic acid (NDGA), which reduces the production of arachidonate metabolites via the lipoxygenase pathway, also reduced basal and TRH or arachidonic-acid-stimulated-PRL release. The inhibitory effect of NDGA on PRL release could be overcome by the addition of 15-HETE. The time course curve of PRL release from cells challenged by 15-HETE had the same profile as that of cells stimulated by TRH. The stimulating effect of 15-HPETE (ED50 = 0.7 x 10(-9) M), which is the direct precursor of 15-HETE, on PRL release was greater than TRH or 15-HETE (ED50 = 6.5 x 10(-9) M). Furthermore 15-HPETE and 15-HETE seemed to affect the release of newly synthesized PRL. These data indicate that 15-HETE and 15-HPETE could be important intracellular components in the control of PRL secretion and may account for at least a part of arachidonate-induced PRL release from GH3.     

5,6-epoxyeicosatrienoic acid, a novel arachidonate metabolite. Mechanism of vasoactivity in the rat

We have reported that 5,6-epoxyeicosatrienoic acid (5,6-EET) was the only cytochrome P-450-dependent arachidonic acid (AA) epoxide to dilate the isolated, perfused caudal artery of the rat. We have investigated the mechanisms by which 5,6-EET dilates the rat-tail artery by studying the effect of deendothelialization and inhibition of AA metabolic pathways (cyclooxygenase, lipoxygenase, and cytochrome P-450 monooxygenase) on the vascular action of the epoxide. Rat isolated caudal arteries were perfused with Krebs-Henseleit solution at 37 degrees C, pH 7.4, and gassed with 95% O2-5% CO2. Arterial tone was elevated with phenylephrine; acetylcholine (0.5 nmol) was used to detect the presence of intact, functional endothelium. Doses of 5,6-EET, from 6.25 to 25.0 nmol, were injected close-arterially. After obtaining control responses, the same doses were randomly retested after deendothelialization or in the presence of inhibitors of AA metabolism. Removal of the endothelium decreased by 70% the vasodilator responses to 5,6-EET. The endothelial dependency was a function of the epoxide interacting with cyclooxygenase of the endothelium, because indomethacin (3 microM) and aspirin (50 microM) prevented the vasodilator response to 5,6-EET while not affecting the response to acetylcholine. SKF-525A (1.1 microM) and metyrapone (150 microM) did not affect the responses to the 5,6-EET, whereas clotrimazole (0.7 microM) and nordihydroguaiaretic acid (2.5 microM) had nonspecific effects, decreasing responses to 5,6-EET and acetylcholine. Because 5,6-EET failed to stimulate detectable release of prostanoids into the effluent from the caudal artery, we conclude that 5,6-EET requires conversion by cyclooxygenase for expression of its vasoactivity.     

Metabolism of arachidonic acid by rat adrenal glomerulosa cells: synthesis of hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids

Metabolites of arachidonic acid have been implicated in the regulation of aldosterone release. To form a basis for further investigations in this area, the present study has isolated and identified the metabolites formed from exogenous arachidonic acid by adrenal zona glomerulosa cells and characterized the effects of several inhibitors on the synthesis of these eicosanoids. Rat adrenal glomerulosa cells metabolized exogenous [14C]arachidonic acid to products comigrating with the prostaglandins (PGs), hydroxyeicosatatraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs). The metabolites were found in the cells and the incubation media; however, none of the metabolites were found esterified to cellular lipids. The major metabolites were identified as 6-keto PGF1 alpha, PGE2, PGF2 alpha, PGD2, 12(S)-HETE, 15(S)-HETE, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET. The identities of the HETEs and EETs were confirmed by gas chromatography/mass spectrometry. There was no evidence for the synthesis of leukotrienes. The cyclooxygenase inhibitor, indomethacin, the lipoxygenase inhibitors, nordihydroguaiaretic acid, baicalein and AA861, and the combined cyclooxygenase/lipoxygenase inhibitors, BW755C and eicosatetrayenoic acid, inhibited the formation of the [14C]PGs, the [14C]HETEs, and the [14C]EETs. Metyrapone and clotrimazole, inhibitors of cytochrome P450, increased the synthesis of [14C]PGs and [14C]HETEs and reduced the synthesis of [14C] EETs. Superoxide dismutase did not alter arachidonic acid metabolism. In contrast, arachidonic acid metabolism was increased in cells pretreated with catalase. These data indicate that adrenal glomerulosa cells metabolize exogenous arachidonic acid to a number of oxygenated metabolites including PGs, HETEs, and EETs. From studies with inhibitors, the EETs appear to be synthesized by a cytochrome P450 epoxygenase and the HETEs by lipoxygenases.     

5-Hydroxyeicosatetraenoic acid increases prolactin release from rat anterior pituitary cells

The enzymatic breakdown of phospholipids to form arachidonic acid and its subsequent conversion to metabolites produced via the lipoxygenase pathway in anterior pituitary cells may contribute to the process of PRL release. The incubation of primary cultures of pituitary cells from female rats with the lipoxygenase product 5-hydroxyeicosatetraenoic acid (5-HETE; 5-100 microM) significantly increased PRL release in a concentration-dependent manner. The release of PRL induced by 45 microM 5-HETE was completely blocked by 1 microM dopamine. Penfluridol, an agent that binds to and inactivates several Ca+2-binding proteins, including calmodulin, decreased (P less than 0.01) basal and 5-HETE-stimulated PRL release. Similarly, 50 microM D-600, a Ca+2 channel antagonist, significantly (P less than 0.01) reduced basal and 5-HETE-induced PRL release. BW755c or RHC 80267, both of which reduce the production of arachidonic acid metabolites, including 5-HETE, significantly reduced basal PRL release. The inhibitory effects of BW755c and RHC 80267 on PRL release, however, could be overcome by the addition of 5-HETE. In conclusion, 5-HETE or similar lipoxygenase metabolites may be important cellular components in the process of PRL release, and the inhibitory action of dopamine on PRL would seem to be mediated at some step after stimulation by these metabolites.     

Involvement of eicosanoids in release of oxytocin and vasopressin from the neural lobe of the rat pituitary

Arachidonic acid (AA) is oxidized via three pathways which result in several series of distinct metabolites. Cyclooxygenase produces prostaglandins (PGs), prostacyclins, and thromboxanes. Lipoxygenase produces hydroperoxy/hydroxyeicosatetraenoic acids (HPETE/HETEs) and leukotrienes. Epoxygenase, a recently uncovered pathway, results in epoxyeicosatrienoic acids (EETs). Based on reverse phase HPLC product analysis, this study establishes that all three pathways of AA metabolism are present in microsomal incubates of the neural lobe of the pituitary gland. Addition of PGE2 to incubated fragments of neural lobes of the rat pituitary stimulates secretion of both arginine vasopressin (AVP) and oxytocin in vitro. Inclusion of 5-HETE and 12-HETE in the incubation medium stimulates marginal release of AVP and oxytocin by 12-HETE only. The magnitude of AVP and oxytocin secretion stimulated by the epoxygenase metabolites 8,9-, 11,12-, and 14,15-EET is equal to that caused by PGE2. Maximal stimulation of secretion (3- to 4-fold) requires an EET concentration 10-15 times greater than that of PGE2. In contrast, 5,6-EET is inactive. These data suggest that oxygenated products of AA play a role in AVP and oxytocin secretion. Although PGs appear to be the dominant arachidonate metabolites involved in the release of AVP and oxytocin, the EETs probably have a contributing role.     

Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites

Treatment of human aortic endothelial cells (EC) with minimally oxidized LDL (or minimally modified LDL, MM-LDL) produces a specific pattern of endothelial cell activation distinct from that produced by LPS, tumor necrosis factor-alpha, and interleukin-1, but similar to other agents that elevate cAMP. The current studies focus on the signal transduction pathways by which MM-LDL activates EC to bind monocytes. We now demonstrate that, in addition to an elevation of cAMP, lipoxygenase products are necessary for the MM-LDL response. Treatment of EC with inhibitors of the lipoxygenase pathway, 5,8,11, 14-eicosatetraynoic acid (ETYA) or cinnamyl-3, 4-dihydroxy-alpha-cyanocinnamate (CDC), blocked monocyte binding in MM-LDL-treated EC (MM-LDL=118+/-13%; MM-LDL+ETYA=33+/-4%; MM-LDL+CDC=23+/-4% increase in monocyte binding) without reducing cAMP levels. To further investigate the role of the lipoxygenase pathway, cellular phospholipids were labeled with arachidonic acid. Treatment of cells for 4 hours with 50 to 100 microg/mL MM-LDL, but not native LDL, caused a 60% increase in arachidonate release into the medium and increased the intracellular formation of 12(S)-HETE (approximately 100% increase). There was little 15(S)-HETE present, and no increase in its levels was observed. We demonstrated that 12(S)-HETE reversed the inhibitory effect of CDC. We also observed a 70% increase in the formation of 11,12-epoxyeicosatrienoic acid (11, 12-EET) in cells treated with MM-LDL. To determine the mechanism of arachidonate release induced by MM-LDL, we examined the effects of MM-LDL on intracellular calcium levels. Treatment of EC with both native LDL and MM-LDL caused a rapid release of intracellular calcium from internal stores. However, several pieces of evidence suggest that calcium release alone does not explain the increased arachidonate release in MM-LDL-treated cells. The present studies suggest that products of 12-lipoxygenase play an important role in MM-LDL action on the induction of monocyte binding to EC.     

Effect of lipoxygenase products on choriogonadotropin secretion by cultured human choriocarcinoma JEG-3 cells pre- and post-stimulation with epidermal growth factor and a phorbol ester

Previous studies using arachidonic acid and preferential inhibitors of the arachidonic acid pathway have implicated the lipoxygenase system in choriogonadotropin (hCG) secretion by JEG-3 cells. Presently, JEG-3 cells are used in order to examine the effect of lipoxygenase products on hCG secretion. Results show that 30 microM 15-hydroxyeicosatetraenoic acid (15-HETE) induces an approximately 3-fold increase in basal hCG secretion, while 5-HETE, 12-HETE, and leukotriene LTA4 have no significant effect. In addition, 15-HETE potentiates the stimulation of hCG secretion induced by 3 nM epidermal growth factor (EGF), but has no significant effect on the stimulation of hCG induced by 22 nM tetradecanoylphorbol acetate (TPA). The present study further implicates the arachidonic acid pathway in the control of hCG secretion and documents that the effect of EGF can be rate-limited by a product of the lipoxygenase system.     

Mechanism of release of beta-endorphin from rat pituitary cells. Role of lipoxygenase products of arachidonic acid

We investigated the effects of metabolites of arachidonic acid on the release of beta-endorphin-like immunoreactivity (beta-end-IR) from rat anterior pituitary cells. Anterior pituitary cells from female rats cultured with arachidonic acid released beta-end-IR in a dose- and time-dependent manner. To determine which metabolites of arachidonic acid stimulated the release of beta-end-IR, we examined the effects of an inhibitor of the cyclooxygenase, indomethacin, and an inhibitor of the 5-lipoxygenase, 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA-861). beta-end-IR release from pituitary cells induced by arachidonic acid was inhibited about 37% by AA-861, but was not affected by indomethacin. Other lipoxygenase inhibitors (eicosatetraynoic and nordihydroguaiaretic acid) also reduced the release of beta-end-IR induced by arachidonic acid. The effects of the 5-lipoxygenase products 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and leukotrienes (LTA4, B4, C4, and D4) on the release of beta-end-IR from rat pituitary cells were also examined. 5-HETE (1-50 microM) elicited a dose-dependent release of beta-end-IR from cultured pituitary cells, and 50 microM 5-HETE induced beta-endorphin release time dependently. LTA4 and LTB4 also significantly stimulated the release of beta-end-IR, but LTC4 and LTD4 had no effect. Other lipoxygenase products (12-hydroxy-5,8,10,14-eicosatetraenoic acid, 12-HETE; 15-hydroxy-5,8,10,14-eicosatetraenoic acid, 15-HETE) were also secretagogues at concentrations of above 1 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased responsiveness of GH3 cells to the dopaminergic inhibition of prolactin

The inhibitory regulation of PRL secretion by dopamine (DA) or the dopaminergic agonists bromergocryptine (CB-154) and apomorphine was studied in cultured GH3 cells, an established rat anterior pituitary cell line which produces both PRL and GH. The basal release of PRL from GH3 cells was unaffected when incubated for 6 h with DA concentrations as high as 10(-4) M. The inability of DA to suppress PRL secretion could not be explained by the catabolism of DA or the presence of unknown inhibitors (e.g. estradiol) in the fetal calf serum present in the incubation media. Apomorphine and CB-154 were only partially effective in suppressing PRL release at high concentrations of 10(-4) and 10(-5) M, respectively. Various concentrations of the dopaminergic antagonist d-butaclamol did not reverse the inhibitory action of 10(-5) M CB-154, while equal concentrations (10(-5) M) of both d- and l-butaclamol significantly suppressed PRL release. The greatly lowered responsiveness of GH3 cells to dopaminergic inhibition of PRL is suggestive of some impairment of DA receptors. This hypothesis is supported by radioligand binding studies in which high affinity dopaminergic binding sites are absent in the same cell line used in this study.     

Evidence supporting a correlation between arachidonic acid release and prolactin secretion from GH3 cells

In this study, pharmacological agents that alter phospholipase A2 activity were examined for their effects on PRL release and arachidonic acid mobilization in GH3 cells, a pituitary tumor cell line. Stimulators of phospholipase A2 activity, melittin and mastoparan, increased PRL release during short term incubation. This stimulation was reduced by carbachol, a cholinergic receptor ligand that inhibits PRL release from GH3 cells. Melittin also caused release of [3H]arachidonic acid that had previously been incorporated into phospholipids. Increased levels of free [3H]arachidonic acid in the medium were associated with a loss of radiolabel from the phospholipid fraction of the cells. The [3H]arachidonic acid in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol was reduced during melittin exposure. In contrast, two inhibitors of phospholipase A2, dibromoacetophenone (BAP) and U10029A, inhibited spontaneous PRL release. BAP also decreased basal release of [3H]arachidonic acid, blocked melitin-induced PRL secretion, and inhibited melittin-induced [3H] arachidonic acid release. Exogenous arachidonic acid at doses from 10 nM to 1 microM stimulated PRL secretion. The phospholipase A2 inhibitor BAP blocked TRH- and vasoactive intestinal peptide-induced PRL release, whereas U10029A blocked cAMP-induced and blunted TRH- and vasoactive intestinal peptide-induced PRL release. The hydrolysis of membrane phospholipids generating free arachidonic acid and lysophospholipid under our experimental conditions correlated with PRL secretion in GH3 cells. Addition of arachidonic acid to the culture medium stimulated PRL secretion. These data suggest that release of arachidonic acid and its subsequent actions may participate in the intracellular regulation of PRL secretion.     

Epoxygenase products of arachidonic acid are endogenous constituents of the hypothalamus involved in D2 receptor-mediated, dopamine-induced release of somatostatin

The epoxyeicosatrienoic acids (EETs) were discovered as products of a cyclooxygenase/lipoxygenase-independent, cytochrome P-450 catalyzed metabolism of arachidonic acid (AA) termed the "epoxygenase" pathway. The rat hypothalamus is able to synthesize EETs from exogenous AA, and 5,6-EET has been found to release the neuropeptide somatostatin (SRIF) from hypothalamic nerve terminals of the median eminence (ME). In the present study, hypothalami from male rats were examined for the presence of endogenous EETs, using chemical, chromatographic, and mass spectral analysis procedures. The samples were initially separated in a C18 Sepralyte column, fractionated on TLC plates, and purified by reverse phase HPLC. Thereafter, they were esterified (pentafluorobenzyl esters) and subjected to negative ion chemical ionization/gas chromatography (GC)/mass spectral (MS) analysis. The GC retention time and the MS fragmentation patterns revealed the presence of a mixture of 8,9-, 11,12- and 14,15-EETs; instability of 5,6-EET during the isolation protocol precluded its identification. Total hypothalamic EET concentration was estimated to be 120 ng/g wet tissue. The 8,9-regiosomer released SRIF from ME nerve terminals with an ED50 of 5 x 10(-12) M; Dopamine (DA) and the D2 receptor agonist PPHT, but not the D1 receptor agonist SKF-38393, induced SRIF release from the ME. This effect was blocked by clotrimazole and ketoconazole, two inhibitors of microsomal cytochrome P-450 function and AA epoxygenase in particular. In contrast, the inhibitors failed to affect the increase in SRIF release induced by 8,9-EET. These results indicate that: 1) in addition to cyclooxygenase and lipoxygenase products, epoxygenase metabolites of AA are endogenous compounds of the hypothalamus, and 2) EETs may mediate the increase in SRIF release from hypothalamic neurons induced by the interaction of DA with D2 receptors.     

The pharmacologic modulation of the release of arachidonic acid metabolites from purified human lung mast cells

Although cyclooxygenase and lipoxygenase inhibitors have been widely used in studies of allergic and inflammatory disorders, the effect of these agents has not been determined on mediators released from purified human lung mast cells. The release of histamine, prostaglandin D2, leukotriene C4, leukotriene B isomers, 5-hydroxyeicosatetraenoic acid (5-HETE), and free arachidonic acid (AA) from preparations of purified human lung mast cells (90 +/- 2% pure) prelabeled with 3H-AA as modified by indomethacin, eicosatriynoic acid (ETI), and diethylcarbamazine (DEC) was determined. Indomethacin (3 microM) markedly (greater than 90%) inhibited the anti-IgE- and ionophore A23187-induced release of PGD2 from these cells but had no effect on the release of histamine or other AA metabolites. Specifically, no increase in LTC production was noted, and no large amount of shunting of products into those produced by lipoxygenases was noted. The ETI, a lipoxygenase inhibitor in many systems, inhibited the release of histamine, PGD2, LTC, LTB isomers, 5-HETE, and free AA, all with an IC50 between 3 and 10 microM. The lack of specificity for lipoxygenase products together with a similar potency for inhibition of histamine release suggests that ETI may act at an early event in the activation process of these cells, apparently prior to the action of phospholipase(s), which releases AA from cellular stores. The DEC inhibited PGD2 release in purified mast cells and histamine release in pure and crude preparations of mast cells, both with IC50 values ranging from 1 to 3 mM. No specificity for enzymes responsible for the production of LTA4 was observed in these cells, unlike the results reported in other systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

12(S)-HETE plays a role as a mediator of expression of platelet CD62 (P-selectin)

The role of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE), an abundant lipoxygenase product from arachidonic acid in platelets, remains unknown. We investigated and characterized the role of 12(S)-HETE in platelet activation. 12(S)-HETE production and CD62 expression were increased by stimulation with thrombin at 0.03 U/ml or higher, while TXB2 synthesis was increased by thrombin at 0.1 U/ml or higher. The platelet 12(S)-HETE production was increased 10 s after stimulation and this was earlier than CD62 expression. The expression of CD62 was inhibited by the lipoxygenase (LOX) inhibitors quercetin and nordihydroguaiaretic acid but was not affected by the cyclooxygenase (COX) inhibitors aspirin and indomethacin. Exogenously added 12(S)-HPETE and 12(S)-HETE enhanced CD62 expression, but other HETEs did not. Consequently, 12-LOX products play a role in the expression of CD62 and could be a second messenger for platelet activation.     

12(S)-HETE plays a role as a mediator of expression of platelet CD62 (P-selectin)

The role of 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE), an abundant lipoxygenase product from arachidonic acid in platelets, remains unknown. We investigated and characterized the role of 12(S)-HETE in platelet activation. 12(S)-HETE production and CD62 expression were increased by stimulation with thrombin at 0.03 U/ml or higher, while TXB2 synthesis was increased by thrombin at 0.1 U/ml or higher. The platelet 12(S)-HETE production was increased 10 s after stimulation and this was earlier than CD62 expression. The expression of CD62 was inhibited by the lipoxygenase (LOX) inhibitors quercetin and nordihydroguaiaretic acid but was not affected by the cyclooxygenase (COX) inhibitors aspirin and indomethacin. Exogenously added 12(S)-HPETE and 12(S)-HETE enhanced CD62 expression, but other HETEs did not. Consequently, 12-LOX products play a role in the expression of CD62 and could be a second messenger for platelet activation.     

Bombesin stimulates prolactin and growth hormone release by pituitary cells in culture

Bombesin (BBS) has been previously shown to stimulate the secretion of PRL and GH in steroid-primed rats. To determine whether these effects were mediated by the central nervous system or were due to direct action on the pituitary gland, we studied the interaction of BBS with GH4C1 cells, a clonal strain of rat pituitary cells which synthesizes and secretes PRL and GH. The addition of 100 nM BBS to GH4C1 cells for 60 min increased PRL release to 140 +/- 3% of the control value (mean +/- SE) and GH release to 133 +/- 5% of the control value. Stimulation of hormone secretion was observed within 15 min of treatment with 100 nM BBS and continued for at least 2 h. Half-maximal stimulation of PRL release occurred with 0.5 nM BBS, and a maximal effect was observed with 10 nM peptide. The BBS analogs ranatensin, litorin, and [Tyr4]BBS, each at a concentration of 100 nM, caused the same stimulation of PRL release as maximal concentrations of BBS itself. BBS stimulated hormone release selectively in two of five different clonal pituitary cell strains examined. Pretreatment of GH4C1 cells with 1 nM estradiol and/or 100 nM insulin resulted in more powerful stimulation of PRL release by both TRH and BBS. When epidermal growth factor and vasoactive intestinal peptide were added simultaneously with BBS, PRL release was greater than in the presence of either peptide alone. In contrast, the stimulatory effects of TRH and BBS were not additive. Somatostatin inhibited both basal and stimulated PRL release. Thus, low concentrations of BBS can directly stimulate PRL and GH release by a clonal pituitary cell strain in culture. These results suggest that BBS may stimulate PRL and GH secretion in vivo by direct action on the pituitary gland.     

Acute stimulated hormone release from cultured GH3 pituitary cells.

Treatment of cultured rat pituitary GH3 cells with 50 mM KCl in growth medium released 33% of cell PRL and 18% of cell GH with a half-time of 5 min. Hormone in the culture medium was increased 2- to 4-fold over unstimulated levels. The response required calcium; barium and strontium, but not magnesium, could substitute for calcium. Low temperature completely inhibited hormone release, which was also reduced significantly by inhibitors of energy metabolism and by nitrogen. This acute response was similar in ionic requirements, hormones released, and time course to the acute effect of TRH. Like potassium stimulation, TRH resulted in acute release of both PRL and GH. This contrasts with the finding that chronic TRH treatment reduced GH synthesis in GH3 cells. After a 10-min preincubation with potassium, subsequent short incubations with potassium released little hormone unless the cells were allowed to recover by incubation in normal medium for at least 2 h. This acutely releasable hormone pool seems to be located in a membrane-bound subcellular fraction, since GH3 cells did not discharge the cytoplasmic marker enzyme, lactate dehydrogenase, during potassium-stimulated hormone release.     

Differential effects of ketoconazole on prolactin and growth hormone release by normal and tumoral rat anterior pituitary cells in vitro

The imidazole derivative ketoconazole (1-100 microM) was shown to stimulate the release of prolactin (PRL) from rat anterior pituitary cells in vitro. In contrast, this drug did not affect growth hormone (GH) release from the same cells. In addition, ketoconazole was found to have no effect on PRL or GH release from a tumoral pituitary cell clone (GH3). Treatment of normal pituitary cells with ketoconazole (10 microM) for more than 20 min abolished TRH-induced hormone release. TRH-stimulated release was both attenuated and delayed in the ketoconazole-treated tumoral cells. Ketoconazole (10 microM) did not affect the basal electrophysiological properties of GH3 cell membranes, although it did affect the TRH-induced response. The action of ketoconazole of the spontaneous release of PRL by normal cells and the TRH-stimulated release of PRL and GH is consistent with an interference with arachidonic acid metabolism.     

Transmembrane signals mediating neural peptide secretion: role of protein kinase C activators and arachidonic acid metabolites in luteinizing hormone-releasing hormone secretion

The present experiments were designed to determine the effects of different activators of protein kinase C on the secretion of LHRH from median eminence nerve terminals incubated in vitro. The release of prostaglandin E2 (PGE2), a metabolite of arachidonic acid intimately involved in the secretion of LHRH, was also evaluated. Synthetic diacylglycerol [1,2-didecanoylglycerol (DiC10)] significantly enhanced PGE2 release in a concentration-dependent manner. Blockade of phospholipase A2 (PLA2) activity nullified this effect. LHRH release, on the other hand, was not increased by DiC10. However, in the presence of a lipoxygenase inhibitor, DiC10 produced a concentration-related increase in LHRH release, which paralleled that in PGE2. Phospholipase C (PLC) increased both PGE2 and LHRH secretion. Again, blockade of the lipoxygenase pathway enhanced the release of LHRH by PLC without affecting the stimulated secretion of PGE2. A phorbol ester, phorbol 12,13-dibutyrate (PDBu), markedly increased LHRH secretion while inducing a modest increase in PGE2 release. Both effects of PDBu were unaffected by lipoxygenase inhibition. DiC10, PDBu, and PLC significantly augmented LHRH secretion from tissues in which metabolism of arachidonic acid had been prevented by inhibition of both cyclooxygenase and lipoxygenase pathways, suggesting that activation of protein kinase C, independent of PLA2 activation, can lead to the secretion of this neural peptide. Some lipoxygenase metabolites had either no effect on [5- and 15-hydroxyeicosatetraenoic (5- and 15-HETE)] or induced a marginal stimulation of (12-HETE) LHRH release. At certain concentrations, 12-HETE enhanced the stimulatory effect of the phorbol ester on LHRH release. Our results suggest that activation of protein kinase C can stimulate LHRH secretion from nerve terminals in vitro and, further, that diacylglycerol may represent an important intracellular messenger participating in the events leading to LHRH secretion. In addition, stimulation with DiC10 and PLC uncovered inhibitory [unknown arachidonic acid metabolite(s) via lipoxygenase] and stimulatory (PGE2 via cyclooxygenase) pathways through with arachidonic acid metabolites may participate in the intracellular transduction of signals modulating neural peptide secretion.     

Incorporation of platelet and leukocyte lipoxygenase metabolites by cultured vascular cells

When arachidonic acid metabolism is studied during platelet-endothelial interactions in vitro, the predominant cyclooxygenase end products of each cell type (thromboxane B2 and 6-keto-prostaglandin-F1 alpha, respectively) are essentially completely recovered in the cell-free supernatants of these reactions. In contrast, 50% of 12-hydroxy- 5,8,10,14-eicosatetraenoic acid (12-HETE), the major lipoxygenase metabolite from platelets, is released into the cell-free supernatant. In investigating the basis of this observation, we have found that platelet lipoxygenase metabolites were generated to the same extent during these coincubations but became rapidly incorporated into the endothelial cells. The endothelial cell-associated 12-HETE was present not only as free fatty acid, but was also incorporated into cellular phospholipids and triglycerides. When purified 3H-12-HETE, 3H-5-HETE (the major hydroxy acid lipoxygenase product of leukocytes), and 3H- arachidonic acid (the common precursor of these metabolites) were individually incubated with suspensions of cultured bovine aortic endothelial cells or smooth muscle cells, different patterns of intracellular lipid distribution were found. In endothelial cells, 12- HETE was incorporated equally into phospholipids and triglycerides, whereas 5-HETE was incorporated preferentially into triglycerides, and arachidonic acid was incorporated into phospholipids. In smooth muscle cells, both 12-HETE and 5-HETE showed more extensive incorporation into triglycerides. The rapid and characteristic incorporation and esterification of platelet and leukocyte monohydroxy fatty acid lipoxygenase products by endothelial and smooth muscle cells suggests a possible physiologic role for these processes in regulating vascular function.     

Synthesis of arachidonic acid-derived lipoxygenase and cytochrome P450 products in the intact human lung vasculature

Lipoxygenase (LO) and cytochrome P450 monooxygenase products of arachidonic acid (AA) have been implicated in a large number of vasoregulatory processes. In intact, blood-free, perfused and ventilated human lungs (n = 8), isolated during surgery for bronchial carcinoma, we analyzed leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs) by sequential sampling of the recirculating buffer fluid. For the analysis we used multistep, solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, with elution of all metabolites within one run and photodiode array detection to obtain full UV spectra of eluting compounds. We detected no LT release in a 15-min baseline period, but the admixture of the calcium ionophore A23187 with the buffer fluid provoked the rapid appearance of all LTs. Some baseline release of 15-HETE was observed, and in response to A23187, maximum buffer concentrations were noted for 5-HETE, with 8-HETE, 9-HETE, 11-HETE, and 12-HETE being detected at lower levels. Marked baseline liberation of 11,12-EET and 8,9-EET was observed. In response to A23187, high oxirane buffer concentrations were registered, which far surpassed those of LTs and HETEs. The eicosanoid release was paralleled by a limited pulmonary artery pressor response and progressive vascular leakage. We conclude that ex-vivo-perfused human lungs release EETs > LTs > HETEs into the vascular compartment in response to inflammatory challenge. The marked oxirane synthesis in the lung vasculature may have major impact on lung vasoregulation when considering the possible function of these AA epoxides as endothelium-derived hyperpolarizing factors.