Kyriaki Gerozissis

Prostanoid profile in specific brain areas, pituitary and pineal gland of the male rat. Influence of experimental conditions.

Using chromatographic separation and specific radioimmunoassays, we were able to detect the prostanoids PGD2, PGF2 alpha, PGE2, 6-keto-PGF1 alpha and thromboxane B2 in selected brain areas, pituitary and pineal glands of adult male rats. Our results suggest regional differences in the biosynthetic capacity of homogenates. Higher amounts were produced by the pituitary and pineal gland (33 and 28 ng/mg protein, respectively). In the hypothalamus, the median eminence was the most active part (24 ng/mg protein). The sum of the 5 prostanoids in whole hypothalamus was higher after 15 min incubation (10 ng/mg protein), than at zero time (2.7 ng) or after immediate freezing of the tissue (1.2 ng). We confirm the predominance of PGD2 after incubation of the homogenates in each of the 8 areas in the brain, pituitary and pineal gland. However, PGD2 was not predominant when the prostanoid profile was estimated immediately after homogenization or when the hypothalamus was immediately frozen in liquid nitrogen. The ratio PGD2/PGF2 alpha was 2.7, after 15 min incubation, 0.9 at zero time and 0.8 for the hypothalamus immediately frozen. Furthermore, PGD2 predominance was not observed when intact tissues were incubated without homogenization.

Leukotrienes C4 and D4 stimulate the release of luteinizing hormone-releasing hormone from rat median eminence in vitro.

The release of luteinizing hormone-releasing hormone (LH-RH), somatostatin (SRIF) and growth releasing factor (GRF) by male rat median eminences (MEs) incubated in vitro for 30 min, in the presence of leukotrienes (LT) C4, D4, E4 and B4 was estimated by radioimmunoassay (RIA). Leukotrienes, with the exception of LTE4 stimulated the release of LH-RH. The dose-response curve was bimodal for LTC4 with two maxima at 10(-8) and 10(-16) M (X2.2 and 1.9, respectively), biphasic for LTD4 with a maximum (X2) at 10(-8) M; LTB4 was active only at 10(-6) M (X1.9). These different curves suggest a specific effect on the release of LH-RH. Moreover, these effects were selective since no alteration of SRIF and GRF secretions was observed. No additive effect on LH-RH release was observed when LTC4 and LTD4 were added simultaneously at 10(-8) M. FPL-55712, a drug supposed to be an antagonist of LTC4, showed an unexpected stimulatory effect (X4.2 and 1.7-fold) on LH-RH release at 3.10(-5) and 10(-6) M, respectively. However, FPL-55712 did not alter the release of LH-RH induced with 10(-8) or 10(-16) M LTC4. These results extend our previous observations on the stimulatory action of LTC4 and are the first evidence of the stimulatory effect of LTD4 and LTB4 on the LH-RH release.

L8027 and 1-nonyl-imidazole as non-selective inhibitors of thromboxane synthesis.

Abstract The inhibition of prostaglandin-related actions on the platelets, respiration and hemodynamics of guinea pigs by 1-(isopropyl-2-indolyl)-3-pyridyl-3-ketone (compound L8027) and by 1-nonyl-imidazole (NI) was studied. L8027 (1–10 μg/kg, i.v.) inhibited arachidonic acid (AA)-initiated bronchoconstriction, thrombocytopenia and hypotension in a dose-dependent and reversible manner. NI, however, in doses as high as 8 mg/kg did not inhibit these actions. AA-induced platelet aggregation was antagonized by L8027 (50–500 nM) and NI (25–100 μM), and this action was dose-dependent and competitive. Thromboxane A2 (TxA2) synthesis in blood and platelets, as measured by the rabbit aorta bioassay, was suppressed in a concentration-related manner by L8027 at anti-aggregatory doses. NI did not inhibit TxA2 synthesis in guinea-pig platelet-rich plasma (PRP) at concentrations which blocked platelet aggregation. However, it did block TxA2 synthesis in rabbit and human PRP and in washed guinea-pig platelets, indicating a species or plasma specificity. Both compounds inhibited ATP release from platelets as they inhibited aggregation. The radioimmunoassay revealed that L8027 decreased formation of both TxB2 and PGE2, unlike imidazole which only blocked formation of TxB2 and resulted in an increase of PGE2. These results show that although L8027 does block TxA2 formation, it is not a selective antagonist and it inhibits cyclo-oxygenase as well. NI appears to have the ability to act via mechanisms other than prostaglandin metabolism.

Leukotriene C4-induced release of LHRH into the hypophyseal portal blood and of LH into the peripheral blood

Intracerebroventricular (i.c.v.) administration of leukotriene (LT) C4 at doses of 2, 0.5 and 0.2 micrograms/rat significantly stimulated (3-12 fold) the release of LH into the peripheral blood of male rats. Injection of anti-LHRH serum had no effect on LTC4-stimulated LH release, but did block PGE2- stimulated LH release. I.c.v.- infused LTC4 also stimulated the release of LHRH into the hypophyseal portal blood. This is the first report of an in vivo action of LTC4 on the release of a hypothalamic releasing factor (LHRH) and a pituitary hormone (LH). These observations, plus in vitro results, clearly show that LTC4 stimulates LH release by acting on both the hypothalamus, causing LHRH release, and on the pituitary. Then the action of LTC4 on LH release in vivo is quite different from the indirect action of PGE2.

Release of luteinizing hormone-releasing hormone: interrelations between eicosanoids and catecholamines

The in vitro release of luteinizing hormone-releasing hormone (LHRH), prostaglandin (PG) E2 and leukotriene (LT) C4 from male rat median eminences (ME), was estimated by radioimmunoassay (RIA) in the presence of the catecholamines (CA), norepinephrine (NE) and dopamine (DA). NE increased the release of PGE2 in the presence and in the absence of the Ca2+ ionophore A23187 (5 x 10(-6) M), but it did not modify the A23187-induced release of LTC4 from endogenous precursors or radiolabelled arachidonic acid. DA also stimulated the A23187-induced release of PGE2 but inhibited that of LTC4. However, while NE increased both the basal and the A23187-induced release of LHRH, DA increased the basal release of LHRH and inhibited the A23187-induced LHRH release. Exogenous LTC4 cancelled the inhibitory effect of DA on LHRH release. Blockade of dopaminergic receptors with haloperidol suppressed the effects of DA on PGE2, LTC4 and LHRH release. Neither eicosanoid affected the K+-evoked [3H]DA release, whereas only PGE2 inhibited the K+-evoked [3H]NE release. We conclude that LTC4 does not interact with the noradrenergic pathway and that the stimulatory effect of both catecholamines on LHRH release involves PGE2, but the inhibitory effect of DA is associated with reduced LTC4 production.

Arachidonate Metabolism in the Anterior Pituitary: Effect of Arachidonate Inhibitors on Basal and Stimulated Secretion of Prolactin, Growth Hormone and Luteinizing Hormone. II. Hormone Release from Dispersed Pituitary Cells

In the accompanying study, we reported the effects of inhibitors of arachidonic acid metabolism on the regulation of prolactin, growth hormone (GH) and luteinizing hormone secretion by male hemipituitaries. The present work extends these investigations to primary cell cultures of the same origin. Arachidonic acid metabolism was inhibited by either 5, 8, 11, 14‐eicosatetraynoic acid (ETYA), a blocker of cyclooxygenase‐ and lipoxygenase‐catalysed pathways, or the cyclooxygenase inhibitors, indomethacin and aspirin.

Prostaglandin E2 and leukotriene C4-induced luteinizing hormone-releasing hormone release from immature and adult male rat median eminence in vitro: Eicosanoid formation and binding parameters

The amounts of prostaglandin E2 formed in vitro by the median eminences of adult male rats were greater than those produced by the median eminences of immature, 22 day-old rats. However, the amount of leukotriene C4 produced by the adult rat median eminences was lower than that produced by the immature rat median eminences. Analysis of the prostaglandin E2 binding parameters of hypothalamic P2 membrane fractions indicates that there are two binding components, one high affinity (RH) and one low affinity (RL) in both adult and immature rats. The maximal binding capacity of RH from adult rat membranes was significantly lower than that of immature rat membranes, correlating with greater prostaglandin E2 production by the adult rat median eminence. Only one leukotriene C4 binding site was detected in both adult and immature rat membranes. Exogenous prostaglandin E2 and leukotriene C4 both stimulated, the release of luteinizing hormone-releasing hormone to the same extent from both the adult and immature median eminences.

Effect of castration on eicosanoid and catecholamine-induced luteinizing hormone-releasing hormone release in vitro.

The castration of adult rats four weeks before experimentation did not alter either the basal production of prostaglandin (PG) E2 (intact rats 1157 +/- 160 pg/mg protein, castrated rats 1093 +/- 90 pg/mg protein) or the A 23187-induced production of PGE2 (1591 +/- 209 or 1701 +/- 286 pg/mg protein respectively) or that of leucotriene (LT) C4 (474 +/- 33, 389 +/- 39 pg/mg protein). Castration significantly reduced the absolute amounts of luteinizing hormone-releasing hormone (LHRH) released under basal conditions (594 +/- 56 pg/mg protein to 34% of intact controls) and after A 23187 stimulation (to 28% of controls). The LHRH released by the median eminence (ME) of castrated rats in the presence of PGE2 (10(-6) M), LTC4 (10(-8) M), norepinephrine (NE) (10(-5) M) or dopamine (DA) (10(-5) M and 10(-4) M) was significantly lower than the LHRH released by intact animals in the presence of these factors. However the relative values for LHRH release (e.g. as % of controls) indicated that the degree of stimulation was identical in intact and castrated rats (2-3 times). These data suggest that the absence of gonadal steroids resulting from long-term castration does not block the stimulatory action of eicosanoids or catecholamines on the release of LHRH by the ME of adult male rats in vitro. Furthermore, castration does not interfere with the biosynthesis of PGE2 and LTC4.