Effect of essential fatty acid deficiency on membrane fatty acid content and growth hormone stimulation of rat pituitaries during postnatal development.

Fatty acid composition of anterior pituitary cell membranes of rats deprived of essential fatty acids (EFA) and of rats receiving a standard diet was determined during postnatal development and in adults. Pregnant rats were fed an EFA-deficient diet and the offspring were fed the same diet after weaning. In parallel, effects of the diet on growth and on growth hormone (GH) responsiveness to GHRH stimulation were determined in control animals. Membrane content of arachidonic acid (20:4n-6) and of its elongation product adrenic acid (22:4n-6) increased regularly from day 2 to day 12 after birth. EFA-deficiency resulted on day 2 in increased oleic acid and in substitution of arachidonic and adrenic acids by corresponding elongation-desaturation products of oleic acid: eicosatrienoic (20:3n-9) and docosatrienoic (22:3n-9) acids. At the age of 24 days, n-9 series fatty acid reached the same level as in adult animals. Two-day-old EFA-deficient rats paradoxically exhibited a higher level of 20:4n-6 as compared to control rats. EFA-deficiency also decreased growth rate and GH pituitary responses to GHRH during the prepubertal period. These results suggest that changes in the lipid structure and in pituitary secretion properties elicited by EFA-deficiency depend upon the stage of development.

Selective effect of a diet-induced decrease in the arachidonic acid membrane-phospholipid content on in vitro phospholipase C and adenylate cyclase-mediated pituitary response to angiotensin II.

Young rats were fed on an essential fatty acid (EFA)-deprived diet for 6 weeks after weaning. Their pituitary was removed and adenohypophyseal cells dispersed and maintained in culture. Membrane lipids were analyzed and basal and stimulated levels of hormone secretion were measured after 4-day incubation in a culture medium containing or not 160 microM arachidonic acid 20:4n-6 (AA) in order to obtain EFA-deficient or EFA-restored pituitary cells, respectively. In EFA-deficient cells membrane phosphoglycerides (PGL) were depleted in AA and adrenic acid 22:4n-6; the deficit was overcome by incubation in the presence of AA. Depletion diversely affected PGL classes. AA was highly depleted in choline phosphoglycerides (ChoPG), only moderately depleted in serine and ethanolamine phosphoglycerides (SerPG and EtnPG) and not depleted at all in inositol phosphoglycerides, suggesting preferential preservation of AA in that class of PGL. Restoration of AA by addition of the fatty acid to the culture medium was complete for ChoPG and EtnPG and only partial for SerPG. Depressed levels of AA and adrenic acid in PGL were compensated for by a concomitant increase in 20:3n-9 and 22:3n-9. Growth hormone and prolactin (PRL) secretion was assessed by radioimmunoassay and possible effects of a membrane AA deficit on hormone regulation were tested in cells challenged by either growth hormone-releasing hormone, thyrotropin-releasing hormone, angiotensin II (AII), vasoactive intestinal peptide (VIP) or dopamine. Neither basal nor stimulated growth hormone secretion was different from controls in EFA-deficient cells. PRL modulation by VIP or dopamine was not affected either in EFA-deficient cells. In contrast, the capacity of AII, but not of thyrotropin-releasing hormone, to release PRL was markedly decreased in EFA-deprived cells. It was restored by addition of AA to the incubation medium. Parallel depression of AII-induced inositol phosphates and cAMP accumulation was also observed after EFA deficiency. When tested on membranes, the paradoxical inhibition of adenylate cyclase by AII documented by previous observations was reinforced in EFA-deficient membranes. In contrast, binding of AII was not affected by EFA deficiency. It is concluded that under our experimental conditions EFA deficiency affects selectively coupling of the AII receptor to its effectors without alteration of binding. The effect could involve changes in receptor interactions with coupling proteins.

Effect of an essential fatty acid deficiency on the phospholipid composition in anterior pituitary membranes.

The effects of an essential fatty acid deficient diet were investigated on the phospholipid fatty acids of several membrane fractions of the rat anterior pituitary, the secretion of which is known to be partly dependent on the membrane phospholipidic constituents. In standard dietary conditions, arachidonic acid (20:4n-6) and its elongation product, adrenic acid (22:4n-6), were the two main polyunsaturated fatty acids in all fractions studied. In rats deprived of EFA for 6 weeks after weaning, the levels of both 20:4n-6 and 22:4n-6 were not changed in microsomal + plasma membrane and nuclear fractions, whereas they were decreased in heavy mitochondrial and light mitochondrial fractions. The present data suggest a mechanism of compensation between membrane fractions which may preferentially preserve 20:4n-6 and 22:4n-6 in discrete membrane fractions.

Arachidonate metabolism in the anterior pituitary: effect of arachidonate inhibitors on Basal and stimulated secretion of prolactin, growth hormone and luteinizing hormone. I. Hormone release from incubated or perifused pituitary fragments.

Abstract The potential involvement of arachidonic acid metabolites in the regulation of adenohypophyseal secretion was analysed on pituitary glands from male rats incubated in the presence of various inhibitors with different mechanisms of action: two inhibitors of phospholipase A(2) (parabromophenacylbromide, PB and compound CB 874), an inhibitor of cyclooxygenase- and lipoxygenase-catalysed pathways (5, 8, 11, 14-eicosatetraynoic acid, ETYA) and an inhibitor of cyclooxygenase (epsilon-lysyl acetylsalicylate, ASP). Under conditions which minimize side effects of the drugs, all inhibitors reduced prostaglandin synthesis and release, without affecting the metabolic integrity of the tissues (assessed by their intracellular adenosine triphosphate levels). All agents tested (PB, ETYA, ASP) suppressed prolactin secretion induced either by thyrotropin-releasing hormone or vasoactive intestinal peptide. Basal prolactin secretion was sensitive to phospholipase A(2) inhibitors. Similar inhibitions were obtained with ETYA and CB 874 on growth hormone secretion under basal conditions as well as after stimulation by growth hormone-releasing factor, thyrotropin-releasing hormone, or vasoactive intestinal peptide. In contrast, luteinizing hormone secretion, stimulated or not by gonadotropin-releasing hormone, was not sensitive to any of the agents used. It is concluded that, in intact male hemipituitaries, arachidonic acid metabolism is involved in the stimulation of prolactin and growth hormone secretion by neuropeptides. In contrast, luteinizing hormone release does not seem to depend on that mechanism. It has been verified that the inhibitors of arachidonic acid metabolism do not directly interfere with adenylate cyclase, or with the activation of protein kinase C, two enzymes which are involved in the regulation of secretory mechanisms.

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.

Abstract 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. ETYA inhibited basal GH secretion by 60%, an effect which was reversed by micromolar concentrations of exogenous arachidonic acid. ETYA was much less effective on growth hormone-releasing factor-induced GH release, a result which contrasts with data obtained on intact glands. Growth hormone-releasing factor stimulation of adenylate cyclase was not affected by ETYA. Cyclooxygenase inhibitors decreased basal secretion to a more limited extent (-30%) and were ineffective on growth hormone-releasing factor-stimulated release. Basal prolactin secretion was reduced by 30% in the presence of ETYA and unaffected by cyclooxygenase inhibitors. As with GH, the effect was reversed by exogenous arachidonic acid. However, in contrast to growth hormone-releasing factor-stimulated GH secretion, thyrotropin-releasing hormone stimulation of prolactin release was able to overcome the inhibition by ETYA in a dose-dependent manner. Again, the insensitivity of thyrotropin-releasing hormone-stimulated prolactin release to ETYA contrasts with the data obtained in intact tissue. Moreover, ETYA inhibited (-60%) prostaglandin E(2) production; thyrotropin-releasing hormone was unable to increase the prostaglandin levels in control or ETYA-treated cells. This confirms the data obtained with cyclooxygenase inhibitors, suggesting that prostaglandins are not involved in prolactin secretion. Intracellular accumulation of Ca(2+) by the ionophore A23187 and protein kinase C stimulation by the phorbol ester 12-O- tetradecanoyl phorbol acetate (TPA), strongly stimulated GH and prolactin release. Under these conditions, ETYA was no longer able to inhibit secretion of the hormones. As with intact glands, basal and gonadotropin-releasing hormone or TPA-induced luteinizing hormone secretion were unaffected by any of the inhibitors used. It is concluded that blockade of the arachidonic acid cascade interferes with a secretory pathway involved mainly with basal release of prolactin and GH, but not luteinizing hormone. Thyrotropin-releasing hormone, a secretagogue known to trigger phospholipase C and, hence, to stimulate Ca(2+) mobilization and protein kinase C, overcame ETYA inhibition of prolactin secretion. Growth hormone-releasing factor, a secretagogue recognized by adenylate cyclase coupled receptors, did not overcome ETYA inhibition of GH secretion. However, both secretagogues strongly stimulated hormone release from their target cells in the presence of ETYA. The arachidonic acid cascade thus seems less important in neuromediator-induced secretion coupling processes in dispersed pituitary cells, than in the intact gland. These observations suggest that eicosanoids are more likely to mediate paracrine or autocrine modulations of secretory mechanisms, rather than to function as intracellular messengers.

Dihydropyridine-sensitive calcium channel activity related to prolactin, growth hormone, and luteinizing hormone release from anterior pituitary cells in culture: interactions with somatostatin, dopamine, and estrogens.

In the present work, we determined the activity of voltage-dependent dihydropyridine (DHP)-sensitive Ca2+ channels related to PRL, GH, and LH secretion in primary cultures of pituitary cells from male or female rats. We investigated their modulation by 17 beta-estradiol (E2) and their involvement in dopamine (DA) and somatostatin (SRIF) inhibition of PRL and GH release. BAY-K-8644 (BAYK), a DHP agonist which increases the opening time of already activated channels, stimulated PRL and GH secretion in a dose-dependent manner. The effect was more pronounced on PRL than on GH release. BAYK-evoked hormone secretion was further amplified by simultaneous application of K+ (30 or 56 mM) to the cell cultures; in parallel, BAYK-induced 45Ca uptake by the cells was potentiated in the presence of depolarizing stimuli. In contrast, BAYK was unable to stimulate LH secretion from male pituitary cells, but it potentiated LHRH- as well as K+-induced LH release; it had only a weak effect on LH secretion from female cell cultures. Basal and BAYK-induced pituitary hormone release were blocked by the Ca2+ channel antagonist nitrendipine. Under no condition did BAYK affect the hydrolysis of phosphoinositides or cAMP formation. Pretreatment of female pituitary cell cultures with E2 (10(-9) M) for 72 h enhanced LH and PRL responses to BAYK, but was ineffective on GH secretion. DA (10(-7) M) inhibited basal and BAYK-induced PRL release from male or female pituitary cells treated or not treated with E2 (10(-9) M). SRIF (10(-9) and 10(-8) M) reversed BAYK-evoked GH release to the same extent in cell cultures derived from male or female animals. It was ineffective on BAYK-induced PRL secretion in the absence of E2, but antagonized it after E2 pretreatment. The effect was dependent upon the time of steroid treatment and was specific, since 17 alpha-estradiol was inactive. In addition, DA and SRIF decreased the 45Ca uptake induced by the calcium agonist. These data demonstrate that DHP-sensitive voltage-dependent calcium channels of the L type present on different pituitary cells are not equally susceptible to BAYK activation under steady state basal conditions, indicating that their spontaneous activity and/or distribution vary according to the cell type; their activity is modulated by sex steroids. In addition, these data suggest that Ca2+ channels represent a possible site of DA and SRIF inhibition of PRL and GH release, respectively, by gating calcium entry into the corresponding cells.

Estradiol activates methylating enzyme(s) involved in the conversion of phosphatidylethanolamine to phosphatidylcholine in rat pituitary membranes.

17 beta-Estradiol (E2) affects the sensitivity of pituitary cells to several neurohormones as LHRH, TRH, or dopamine, presumably by modulating receptor coupling mechanisms. We attempted to pinpoint the membrane processes underlying this modulation and studied the effect of E2 on pituitary membrane phospholipid methylation. Anterior pituitary membranes prepared from ovariectomized (ovx) or ovx plus E2-treated rats were assayed for phospholipid methylation. Methylated phospholipids were separated by TLC. Incorporation of [3H]methyl groups into phospholipids increased with membrane concentration and incubation time with S-adenosyl-L-methyl [3H]methionine; it was not Mg2+ dependent and was inhibited in a dose-dependent manner by S-adenosyl-L-homocysteine, methyltransferase inhibitor. pH was found to be critical. Formation of phosphatidyl-monoethanolamine, phosphatidyl-dimethylethanolamine, and phosphatidylcholine was markedly stimulated by treatment with E2. The effect increased progressively when animals were killed 15 h to 5 days after E2 implantation. The response involved a shift in the maximum velocity (Vmax) although there was no change in the available substrate for the methylating enzyme. This change in Vmax probably reflects changes in the amount of the methylating enzyme itself. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2 was ineffective, pointing to a stereospecific interaction. After differential centrifugation of pituitary membranes, the highest specific methyltransferase activity was found in light mitochondrial (L) and microsomal (P) fractions and the lowest in nuclei (N) and the heavy mitochondrial (M) fractions. After sucrose density gradient centrifugation, methylated phospholipids were preferentially recovered from fractions corresponding to the endoplasmic reticulum and/or secretory granules. E2 treatment for 5 days did not modify the subcellular distribution of methyltransferase activity but stimulated it in all fractions; in contrast, it did not modify the activity of the other enzymes measured as fraction markers. Under the same experimental conditions, phospholipid methylation in membranes prepared from cortex, and anterior and mediobasal hypothalamic structures was not affected by the steroid, with the exception of a slight increment of [3H]methyl incorporation into mediobasal hypothalamic membrane phospholipids after 5 days of E2 treatment. These results indicate that E2-induced changes in pituitary responsiveness might be concomitant with selective effects of the steroid on specific membrane enzymatic activities involved in coupling mechanisms.

Reutilization of insulin receptor and hormonal response in cultured foetal hepatocytes: the effects of chloroquine and vinblastine.

The effects of chloroquine and vinblastine (10-100 microM) on insulin degradation and biological action were studied in cultured foetal rat hepatocytes. Insulin degradation, as measured by the release of trichloroacetic acid-soluble radioactivity from 125I-insulin into the medium, was strictly cell-associated, saturable with respect to insulin concentrations and linearly related to the amount of cell-associated hormone. The maximal rate of insulin degradation was 4,700 molecules/min per cell, and its KM about 5 nM. Thus, insulin receptors (30,000 sites/cell; half-life close to 13 hr) must be reutilized 450-fold before being degraded with an average time of reutilization inferior to 10 min. In the presence of 70 microM chloroquine or 100 microM vinblastine, insulin degradation was inhibited by 80% and the amount of cell-associated hormone enhanced 2-3-fold. Nearly total inhibition of insulin-stimulated glycogenesis was obtained with 70 microM chloroquine and 45 microM vinblastine. When hepatocytes were preincubated with chloroquine or vinblastine, insulin binding remained high for up to 4 hr, then progressively decreased thereafter. The addition of 10 nM native insulin during preincubation with the drugs resulted in an earlier and more pronounced decrease in insulin binding, whereas native insulin alone did not induce any change. Both the inhibition of insulin degradation and onset of receptor down-regulation suggest a drug-induced impairment in the receptor reutilization. This defect is correlated to a loss of the glycogenic effect of insulin in cultured foetal rat hepatocytes.

[Role of lysosomes, microtubules and microfilaments in the mechanism of the lactogenic action of prolactin in the rabbit mammary gland]. / Rôle des lysosomes, des microtubules et des microfilaments dans le mécanisme de l'action lactogène de la prolactine sur la glande mammaire de Lapine.

The lysosomotropic agents, ammonium chloride and chloroquine, added to the culture medium of pseudopregnant Rabbit mammary gland, did not inhibit the initiation of casein synthesis by prolactin. By contrast, they considerably reduced the down-regulation of prolacting receptors. Converse-y, colchicine totally blocked the lactogenic action of prolactin without altering the down-regulation of the receptor. Cytochalasin B inhibited only partly the lactogenic action of prolactin while it has no effect on the down-regulation of the receptor. These data suggest that the degradation of the prolactin-receptor complex in lysosome is not a compulsory step in the mechanism of prolactin action. The integrity of microtubules but not of microfilaments is required for prolactin to initiate casein synthesis. These elements of the cytoskeleton are not strictly involved in the down-regulation of the receptor.

The combined action of insulin and phlorizin on transport and metabolism of sugars and nucleotide turnover in the isolated rat diaphragm.

The combined effects of insulin and phlorizin have been analyzed on two parameters of insulin stimulation in the surviving rat diaphragm : transport and metabolism of sugars, turnover of the phosphate groups of mononucleotides. Phlorizin (c mM) inhibits glucose transport both in the presence and absence of insulin and displays a small additional inhibitory effect on glycogen biosynthesis; with the non metabolizable glucose anlogue 3-O-methyl-D-glucose transport inhibition is demonstrated solely in the presence of insulin. No correlation is demonstrated between the rates of sugar transport, which are strongly phlorizin-sensitive, and the rates of nucleotide turnover, which show no such sensitivity.

Biosynthesis of acid mucopolysaccharides by the surviving new born rat skin. I. - Kinetics of the biosynthesis at the polymer level.

1) The amounts of individual mucopolysaccharides in the new born rat skin have been estimated and their specific rates of labelling assessed in vitro. Total and percentage amounts of these polymers agree satisfactorily with previously published data. 2) Relative rates of labelling from [U14C]-glucose have been estimated by combining column chromatography separation and electrophoresis on cellulose acetate strips. Specific radioactivities have been measured either with respect to the total uronic acid content of the fractions or with respect to their quantitative staining with Alcian Blue. The two methods agreed satisfactorily. 3) Average biosynthetic rates almost identical for hyaluronic acid and the total sulfated mucopolysaccharides. However, within the latter fraction, heparin + heparan sulfate incorporate [U14C]-glucose about 4 to 5 times more rapidly than the chondroitin sulfates. This result could not be expected from previous data obtained in vivo and is discussed with reference to a possible heterogeneity of the cell material whence the various mucopolysaccharides originate. 4) In the presence of puromycin, labelling of the sulfated mucopolysaccharides stops almost immediately, indicating a stringent requirement for protein primers. Biosynthesis of hyaluronic acid is affected only after preincubation of tissue with puromycin (one hour) and subsequent incubation of two hours with [U14C]-glucose.

Biosynthesis of acid mucopolysaccharides by the surviving new born rat skin. II. - Specific labelling of individual monosaccharides from (U14C)-glucose. Metabolic origin of L-iduronic acid.

1)Individual monosaccharides (uronic acids and aminosugars) have been purified following specific hydrolysis of the mucopolysaccharides from new born rat skin (hyaluronic acid, heparin + heparan sulfate, chondroitin sulfate A, B and C), after incubation with [U14C]-glucose under various conditions and for varying incubation periods. The yields and the specificity of the methods used for hydrolysis are discussed. 2) Monosaccharides from hyaluronic acid and the sulfated mucopolysaccharide fraction are labelled at an approximately equal rate. In addition, high rates of labelling of glucosamine isolated from the sulfated fractions confirms the preferential labelling of (heparin + heparan sulfate) demonstrated with the sulfated polymers. 3) In all fractions, aminosugars are considerably less labelled than the corresponding uronic acids, which suggests the existence of endogeneous diluting precursor pools for the former monosaccharides. 4) No drift of radioactivity from D-glucuronate to L-iduronate could be demonstrated in sulfated mucopolysaccharides after inhibition of their biosynthesis by puromycin or diluting the labelled precursor pools. Hence it has not been possible to substantiate on the surviving tissue the C5 epimerization at the polymer level, as previously demonstrated by other authors with subcellular fractions of various origin.