Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine.

The time-course incorporation of 10 microM [14C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [14C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [14C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [14C]AA and PC = PE for [14C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 microM [3H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 microM, except for a lower proportion in TAG. The stimulation of 3H-labeled cells by 100 microM norepinephrine induced a 170% increase of basal release of [3H]AA into the medium, while [3H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [3H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.

Endothelin stimulates steroid secretion by frog adrenal gland in vitro: evidence for the involvement of prostaglandins and extracellular calcium in the mechanism of actin of endothelin.

Endothelin (ET-1) is a pleiotropic regulatory peptide which exerts multiple endocrine actions on the cardiovascular system. In the present study, we have investigated the possible role of ET-1 in the regulation of adrenocortical cells using perifused frog interrenal (adrenal) slices. Graded doses of ET-1 from 10(-11)-10(-9) M stimulated both corticosterone and aldosterone production in a dose-dependent manner. Repeated 20-min pulses of ET-1 (10(-9) M), given at the frequency of one pulse per 90 min, resulted in a marked reduction of the secretory response after the second pulse. Prolonged administration (3 h) of ET-1 induced a rapid increase in corticosterone and aldosterone output, followed by a gradual decline of corticosteroid secretion. Perifusion of frog adrenal tissue with ET-1 (10(-9) M) caused a significant increase in the release of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable metabolite of the prostacyclin PGI2. The enhancement of PG biosynthesis preceded by 10 min the peak of corticosteroids. When repeated pulses of ET-1 were administered, a significant diminution of the production of PGE2 and 6-keto-PGF1 alpha was observed after the second pulse. The cyclooxygenase inhibitor indomethacin (5 microM) totally suppressed the stimulatory effect of ET-1 on corticosterone and aldosterone secretion; in contrast, indomethacin did not affect ACTH-evoked corticosteroid secretion. Perifusion of adrenal slices with a calcium-free solution or addition of cobalt (4 mM) induced total inhibition of the stimulatory effect of ET-1. These results demonstrate that ET-1 is a potent stimulator of corticosterone and aldosterone secretion from frog adrenal gland in vitro. Our data show that repeated or prolonged administration of ET-1 induces a rapid desensitization phenomenon. The data also indicate that ET-1-evoked corticosteroid secretion is mediated by an increase in PG biosynthesis and requires the presence of extracellular calcium.

The habituation of brainstem catecholaminergic groups to chronic daily restraint stress is stress specific like that of the hypothalamo-pituitary-adrenal axis.

It has previously been shown that immobilization and ether stress induce activation of the hypothalamo-pituitary-adrenal (HPA) axis and that this activation occurs subsequent to activation of brain stem catecholaminergic neurones. In the present study we have investigated whether the brain stem catecholaminergic (CA) neurons show habituation to chronic daily intermittent exposure to the same restraint stress comparable to that of the HPA axis. The level of activity of the brainstem CA groups was estimated by measurement in tissue punches of content of 3,4-dihydroxyphenylacetic acid (DOPAC), a side metabolite of noradrenaline and adrenaline biosynthesis which has been shown to be a reliable index of the stress-induced activation of the CA groups. The level of activity of the HPA axis was determined by measurement of plasma corticosterone and adrenocorticotropic hormone (ACTH) levels. The animals were submitted to a 15 min restraint stress daily. They were sacrificed at the end of the stress session on day 3, 5 and 10. The ACTH response to the acute restraint stress whilst unchanged on day 3 was significantly decreased on day 5 (-54%) and day 10 (-70%) compared to the response in naive rats. The approximately twofold increase in DOPAC level induced by acute restraint stress in the so-called CA medullary group A1/C1 of naive rats was reduced in daily restraint rats on day 5 (-22%) and day 10 (-30%) but was unchanged on day 3. A small (-20%) decrease of the stress-induced DOPAC response in the A2/C2 CA group and locus coeruleus was also observed on day 10.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for adenosine A2b receptors in the rat pineal gland.

We describe the effects of 5'-N-ethylcarboxamidoadenosine (NECA), a mixed A2a/A2b adenosine receptor agonist and 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoaden osin e (CGS 21680), a selective A2a agonist, on cyclic AMP and N-acetylserotonin synthesis in rat pineal gland. NECA, 1 and 10 microM, increased cyclic AMP by 5- and 25-fold and N-acetylserotonin by 40- and 60-fold respectively, whereas CGS 21680 at the same concentrations was ineffective. These results argue for the presence of adenosine A2b receptors in rat pinealocytes.

Formation, metabolism, and action of hepoxilin A3 in the rat pineal gland.

The present study was undertaken to investigate the possible formation of hepoxilin A3 in the rat pineal gland and to study the potential physiological role for this compound in this tissue. Incubation of homogenates of rat pineal glands with arachidonic acid (66 microM) led to the appearance of hepoxilin A3 (HxA3) analyzed as its stable trihydroxy derivative, trioxilin A3 by gas chromatography in both the electron impact and negative ion chemical ionization modes. Endogenous formation of HxA3 is estimated to be 1.43 +/- 0.66 ng/micrograms of protein. This amount is not modified when the tissue is boiled (2.07 +/- 0.66 ng/micrograms of protein). However, the formation of this compound was stimulated to 21.26 +/- 5.82 ng/micrograms of protein when exogenous arachidonic acid was added to the homogenate. Addition of the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (10 micrograms) resulted in a partial blockade of hepoxilin formation. Using [1-14C]HxA3, we demonstrated that the pineal gland contained hepoxilin epoxide hydrolase, which hydrolyzed HxA3 into trioxilin A3. This hydrolysis was inhibited by 1 mumol/L of 3,3,3-trichloropropene-1,2-oxide. In a separate study, HxA3 in the presence of 3,3,3-trichloropropene-1,2-oxide to block the hydrolysis of HxA3 decreased the production of cyclic AMP in cultured organ rat pineals after stimulation with 5'-N-ethylcarboxamidoadenosine, an A1/A2 adenosine receptor agonist. This effect is stereospecific because the (8S)-enantiomer is more active in decreasing cyclic AMP production (-88.7%) than the (8R)-enantiomer. This is the first demonstration of the presence, metabolism, and action of HxA3 in the rat pineal gland.

Effect of an n-3 fatty acid-deficient diet on the adenosine-dependent melatonin release in cultured rat pineal.

We studied the effect of a diet deficient in n-3 fatty acids on the adenosine-dependent melatonin release from cultured rat pineal gland after stimulation by 5'-N-ethylcarboxamidoadenosine (NECA), an A2 adenosine agonist. Experiments were conducted with 2-month-old rats raised on semipurified diets containing either peanut oil (n-3 deficients) or peanut plus rapeseed oil (controls). The proportion of docosahexaenoic acid (22:6 n-3) in the pineal total lipid fraction and in phosphatidylcholine and phosphatidylethanolamine was significantly decreased in n-3-deficient rats. This was compensated for partially by an increase in 22:4 n-6 and 22:5 n-6 levels. The activity of the cultured rat pineal, in terms of cyclic AMP content and N-acetylserotonin and melatonin release in the medium, was lower after stimulation by 10(-5) mol/L NECA in the group fed peanut oil than in the group fed peanut plus rapeseed oil. The increased ratio of n-6/n-3 fatty acids in pineal total lipids and the major glycerophospholipids (phosphatidylcholine and phosphatidylethanolamine) may have an important influence on the rat pineal responses. The results are discussed in the context of changes in membrane-bound proteins, including enzymes and/or receptors involved in the rat pineal gland function.