Stimulation of ovine placental lactogen secretion by arachidonic acid

To determine whether arachidonic acid stimulates the secretion of ovine placental lactogen (oPL), arachidonic acid was infused as an intravenous bolus into pregnant ewes and fetuses. Plasma oPL concentrations were determined in mothers and fetuses before and for 5 h after infusion. The administration of 12.5 mg arachidonic acid (0.15-0.2 mg/kg, n = 11 experiments) to the pregnant ewes caused an increase in maternal plasma oPL concentrations of 73.9 +/- 15.6% (S.E.M.) and 60.8 +/- 18.1% above the pretreatment concentrations at 4 and 5 h respectively (P less than 0.01 in each instance). The infusion of 25 mg arachidonic acid (n = 8) caused increases of 96.0 +/- 19.1% and 100.3 +/- 26.4% (P less than 0.005), and the stimulation was not inhibited by the cyclo-oxygenase inhibitors indomethacin and ibuprofen. In contrast to arachidonic acid, vehicle alone or palmitic acid had no effects on plasma oPL concentrations. Despite the increase in maternal plasma oPL concentrations, plasma oPL concentrations in the fetus remained unchanged after the maternal infusions. The infusion of arachidonic acid (0.5-1.5 mg/kg) directly into six fetuses had no effects on either fetal or maternal oPL concentrations. These studies indicate that arachidonic acid stimulates maternal plasma oPL concentrations but has no effect on fetal oPL concentrations and the stimulation of oPL secretion is not due to the conversion of arachidonic acid to prostaglandins or other cyclo-oxygenase products.     

Interaction between purified ovine inhibin and steroids on the release of gonadotropins from cultured rat pituitary cells

Primary pituitary cell cultures derived from adult male rats were used to explore the interaction between purified 32K ovine inhibin and gonadal steroids on the modulation of basal and GnRH-stimulated secretion of FSH and LH. Purified o-inhibin was a potent inhibitor of basal FSH secretion (IC50 = 3.4 pM) and 10 nM GnRH-stimulated FSH and LH secretion (IC50 = 8.8 and 18.4 pM, respectively). Purified o-inhibin had no effect on basal LH secretion. In this system, the androgens testosterone, 5 alpha-dihydrotestosterone, and androstenedione slightly stimulated basal FSH secretion, and o-inhibin could reverse this stimulatory effect. By contrast, androgens suppress GnRH-stimulated FSH and LH secretion. In combination, androgens and o-inhibin provided greater inhibition of GnRH-mediated gonadotropin secretion than either did alone.     

Gonadotrophin surge-attenuating factor attenuates in-vitro LH secretion induced by gonadotrophin-releasing hormone from cultured ovine pituitary cells only during the breeding season.

Primary cultures of ovine pituitaries from adult ewes were used to investigate aspects of gonadotrophin surge-attenuating factor (GnSAF) bioactivity in human follicular fluid (hFF) from superovulated women. During the autumn and first half of the winter, LH secretion induced by gonadotrophin-releasing hormone (GnRH) was markedly reduced (43.5 +/- 5.2% of control GnRH-induced LH secretion) by incubation for 48 h with steroid-free hFF. For the rest of the year, treatment with the same batch of steroid-free hFF resulted in non-significant reduction or stimulation of GnRH-induced LH secretion (71.3 +/- 13.2 to 117.8 +/- 11.2% of control GnRH-induced LH secretion). Incubation of pituitary cells for 48 h with oestradiol (1 pmol/l to 1 mumol/l), progesterone (1 pmol/l to 1 mumol/l) or oestradiol and progesterone combined (1 pmol/l to 1 mumol/l) in a two-way titration for 48 h had no significant effect on GnRH-induced LH secretion (83.4 +/- 7.6 to 110.6 +/- 5.0% of control secretion). Separating hFF into fractions of different molecular mass by ultrafiltration demonstrated that GnSAF bioactivity was present in a form 10-30 kDa in size. Incubation for 48 h with these fractions had no significant effect on basal FSH secretion but significantly attenuated GnRH-induced LH secretion during the autumn. The same fractions had little effect on GnRH-induced LH secretion from pituitary cells collected during the summer. We conclude that ovine pituitaries display at least partial reduction in sensitivity to GnSAF outside the breeding season.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inverse effects of activin and inhibin on the synthesis and secretion of FSH and LH by ovine pituitary cells in vitro

Little information is available on the effects of activin and inhibin on the synthesis and secretion of pituitary gonadotrophins in species other than the rat. In this in-vitro study, ovine pituitary cell cultures derived from immature sheep were used to investigate the effects of recombinant human activin-A and native Mr 32,000 bovine inhibin on basal and gonadotrophin-releasing hormone (GnRH)-induced release of FSH and LH. Residual cellular contents of FSH and LH were also determined, allowing total content/well to be calculated. Activin-A promoted a dose-dependent increase in basal (+72%; P less than 0.001) and GnRH-induced (+25%; P less than 0.001) release of FSH as well as in the residual cell content (+114%; P less than 0.001) and total FSH content/well (+67%; P less than 0.001). Conversely, inhibin significantly (P less than 0.001) suppressed each aspect of FSH production examined, confirming that in sheep, as in rats, activin and inhibin exert opposing effects on pituitary FSH production. In contrast to the rat, however, in which activin is reported to have no effect on LH secretion, exposure of sheep pituitary cells to activin-A promoted a dose-dependent suppression (-42%; P less than 0.001) of GnRH-induced LH release. This was associated with a corresponding increase (P less than 0.001) in residual cellular content of LH. Consistent with a previous report from this laboratory, inhibin had the opposite effect and significantly enhanced (+47%; P less than 0.001) GnRH-induced LH release. This was associated with a corresponding fall (P less than 0.01) in residual cellular content of LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of human LH isohormones from fresh pituitary tissue

The pattern of human LH (hLH) microheterogeneity was determined using freshly obtained pituitary tissue. Chromatofocusing across a pH 9-6 gradient produced several, distinct peaks of hLH immunoreactivity between pH 8.5 and 6.8, as well as a 'salt peak'. Chromatofocusing of the 'salt peak' across a pH 7-4 gradient yielded distinct peaks of hLH at pH 6.1, 5.6, and 5.4. Pituitary tissue obtained at surgery produced a virtually identical pattern. By gel filtration the elution volume of each major chromatofocusing peak was similar to that of intact hLH, rather than those of the hLH subunits. The bioactivity/immunoreactivity ratios of the major chromatofocusing peaks fell dramatically with decreasing pH, from approximately 8 at pH 8.5 to approximately 1 at pH less than 6. These results indicate that human pituitary LH exists in multiple, isomeric forms that differ markedly in charge and bioactivity. These differences appear to be inherent in the native, intact molecules and not the result of autolysis or of dissociation into subunits.     

Role of arachidonic acid metabolism in ACTH-stimulated cortisol secretion by bovine adrenocortical cells

We have studied the effects of inhibitors of arachidonic acid (AA) metabolism, nordihydroguaiaretic acid (NDGA), a lipoxygenase (LPX) inhibitor, and indomethacin (INDO), a cyclooxygenase (COX) inhibitor, on cortisol secretion and StAR protein in primary cultures of bovine adrenal zona fasciculata (ZF) cells. NDGA inhibited cortisol secretion in response to both 10(-12) M and 10(-8) M ACTH. AA (10(-4) M) partially reversed the inhibition of cortisol secretion by NDGA at 10(-12) M ACTH but not at 10(-8) M ACTH. On the other hand, INDO potentiated the cortisol response to 10(-12) M ACTH. Neither NDGA nor INDO significantly affected StAR protein levels. These results suggest a StAR protein-independent role for the LPX and COX pathways in acute cortisol secretion, and support the hypothesis that LPX products of AA metabolism are key cellular signals when bovine ZF cells are acutely stimulated by physiological concentrations of ACTH (10(-12) M).     

In vitro effect of ethanol exposure on basal and GnRH-stimulated LH secretion from pituitary cells

The question of whether ethanol's (ETOH's) known suppressive effect on serum luteinizing hormone (LH) could be mediated directly at the anterior pituitary level was addressed by examining the effects of ETOH in vitro on release of LH from cultured male rat pituitary cells. The impact of added ethanol concentrations ranging from 50 to 400 mg% on LH release was examined in the basal state and after stimulation by gonadotropin-releasing hormone (GnRH) at a dose of 5 x 10(-10) M. While ETOH did not significantly suppress basal LH release, secretion stimulated with GnRH was noted to be attenuated with higher doses of ETOH (greater than or equal to 100 mg%) compared to stimulated control cells. It is concluded that ETOH exposure in vitro alters stimulated LH secretion by acting directly on pituitary gonadotropes.     

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.     

Effects of leptin on secretion of LH and FSH from primary cultured female rat pituitary cells

BACKGROUND: Leptin, which is the product of the obese gene, is believed to play important roles in pubertal development and reproductive function in females. In a study using adult male rats, it was found that leptin stimulated secretion of gonadotropin from the pituitary in a dose-related manner. However, there has been no such study in female rats. OBJECTIVE: To investigate the effects of leptin on the production of LH and FSH from the pituitary in female rats, using primary cultured pituitary cells. METHODS: In this study, we determined body weight, serum leptin concentration and serum estradiol (E(2)) concentration in female Wistar rats at 3, 5, 6, 7, 9 and 11 weeks of age, and cultured pituitary cells from 6-week-old female Wistar rats with leptin (0--10(-7) mol/l) and GnRH (0 or 10(-8) mol/l). Then basal and GnRH-stimulated extra- and intracellular LH and FSH were assayed by RIA. RESULTS: Serum leptin concentration increased with increases in body weight and E(2) concentration. The pubertal serum leptin concentration was about 10(-10) mol/l. At a lower or moderate concentration, leptin produced dose-related increases in both basal and GnRH-stimulated extra- and intracellular LH and FSH in pituitary cells. At a concentration of 10 mol/l, leptin significantly (P<0.05) stimulated both basal and GnRH-stimulated extra- and intracellular LH and FSH. However, at greater concentrations, these effects diminished. CONCLUSIONS: These results indicated that leptin induced pituitary cells to produce and secrete both LH and FSH, with or without GnRH. The concentration of leptin that induced the greatest production of gonadotropins by pituitary cells was 10(-10) mol/l, which was the same as the physiological pubertal concentration. Leptin may be involved in the onset of puberty. It is also conceivable that leptin may be a cause of ovulatory failure, not only in weight loss but also in weight gain.     

Control of oxytocin secretion by ovine corpora lutea: effects of arachidonic acid, phospholipases, and prostaglandins

The involvement of arachidonic acid and arachidonic acid metabolites in the control of oxytocin secretion by ovine corpus luteum was investigated, using slices of luteal tissue incubated in vitro. Oxytocin was secreted at steady rates by luteal slices, during 60-min incubations (315.0 +/- 45.3 pg/mg.h). The secretion of oxytocin was stimulated by arachidonic acid, phospholipase A2 (PLA2), and phospholipase C (PLC) in a dose-dependent manner. The highest doses of arachidonic acid, PLA2, and PLC used stimulated oxytocin secretion by 145.8 +/- 23.0% (P less than 0.01; n = 6), 331.5 +/- 42.4% (P less than 0.02; n = 4), and 955.5 +/- 278.6% (P less than 0.01; n = 4), respectively. Oxytocin secretion by luteal slices was not affected by either prostaglandin F2 alpha (PGF2 alpha) or PGE2 over a concentration range from 3-3000 nM. Furthermore, inhibitors of the cyclo-oxygenase pathway of arachidonic acid metabolism did not consistently affect arachidonic acid and PLA2-stimulated oxytocin secretion. Nordihydroguaiaretic acid, which inhibits 5-lipoxygenase, however, totally abolished arachidonic acid- and reduced PLA2-stimulated oxytocin secretion. The presence of CoCl2 in the incubation medium also significantly reduced basal and PLA2- and PLC-stimulated oxytocin secretion [P less than 0.05 (n = 5), P less than 0.05 (n = 5), and P less than 0.01 (n = 6), respectively]. We have shown that oxytocin secretion from slices of ovine corpus luteum incubated in vitro is stimulated by exogenous and endogenously released arachidonic acid. The data show that PGF2 alpha and PGE2 do not have a role in luteal oxytocin secretion in vitro and PG formation does not appear to be involved in the stimulation of oxytocin secretion elicited by arachidonic acid or PLA2. Arachidonic acid may have its effect via the lipoxygenase pathway.     

IGF-Ⅰ,胰岛素和雌二醇对雌性大鼠垂体细胞分泌LH的影响

LH分泌受到性激素正、负反馈调节的控制.胰岛素样生长因子-Ⅰ(IGF-Ⅰ)可能也参与LH分泌的调节[1].本实验观察在有或无血清的培养条件下,IGF-Ⅰ和E2对雌性大鼠垂体细胞LH分泌的影响.     

The use of mouse pituitary fragments to study LH secretion

This work evaluated a perifusion system for studying LH secretion from the anterior pituitary (AP) of female mice. Pituitary fragments were challenged with LHRH, and the effluents assayed for LH. In general, the tissue exhibited augmented release to repeated stimulation. In the dose-response study, the amount of LHRH required to produce maximum and half maximum responses dropped almost 10 fold by the 3rd stimulus. In response to various pulse frequencies LH release increased with the frequency of the 100 nM LHRH dose, but the tissue became refractive to constant nonpulsatile stimulation. Other preparations, subjected to high-frequency 10 nM LHRH pulses, released LH in two distinct episodes. All but the first hour of the response was blocked by cycloheximide, confirming the role of protein synthesis in the sustained release of LH. By varying both the pulse frequency and amplitude, a LHRH protocol was found that produced a proestrous-like surge. Lastly, rat and mouse tissues responded similarly to pulsatile LHRH, verifying their similar LH function during the preovulatory period. These studies demonstrate that the perifusion technique can be used for studying LH secretion in the mouse. Its application to other mouse-oriented studies is planned.     

Negative feedback regulation of adrenocorticotropin secretion by cortisol in ovine fetuses

The purpose of this study was to test the hypothesis that physiological increases in the fetal plasma cortisol concentration inhibit fetal ACTH responses to stress. Fetal sheep, between 121 and 131 days gestation, were infused with cortisol (4 micrograms/min) or vehicle for 5 h. One hour after the end of the cortisol or vehicle infusion, fetuses were infused with sodium nitroprusside (100 micrograms/min) to stimulate fetal ACTH and adrenal corticosteroid secretion. Cortisol, but not vehicle, elevated fetal plasma cortisol and suppressed the fetal ACTH and cortisol responses to nitroprusside. Cortisol and 11-deoxycortisol concentrations were significantly correlated in fetal plasma samples drawn during experiments in which cortisol was not infused; however, the cortisol to 11-deoxycortisol ratio was significantly increased during the infusion of nitroprusside. Fetal heart rate increased during vehicle infusion and decreased during cortisol infusion. Fetal blood pressure was not altered by either cortisol or vehicle infusion. Cortisol infusion increased fetal blood hemoglobin concentration, decreased maternal blood hemoglobin concentration, and produced metabolic acidosis in both mother and fetus. Vehicle infusion did not alter either fetal or maternal hemoglobin or pH. The data do not suggest an obvious mechanism for the cortisol-induced changes in fetal and maternal pH and hemoglobin or in fetal heart rate. However, some of the changes might be attributable to changes in fetal sympathetic outflow or to fluid shifts. We conclude that physiological increases in fetal plasma cortisol concentration: 1) inhibit subsequent ACTH responses to stress and 2) alter fetal cardiovascular function.     

Comparison of tests of stress-released cortisol secretion in pituitary disease

OBJECTIVES We wished to compare peak and incremental rise in plasma cortisol in response to insulin induced hypoglycaemia (IIH) stress test, i.m. glucagon stimulation test (IMGST) and short Synacthen test (SST) in patients with pituitary disease, using a modern radioimmunoassay for cortisol. We compared the three stimulants using receiver operator characteristic (ROC) plots, assuming a cortisol threshold of 500 nmol/l or 580 nmol/l for the IIH stress test which we used as the standard from which to evaluate the SST and the IMGST. PATIENTS AND DESIGN We prospectively studied 16 patients (8F, 8M mean age 43.69 ± 3.72 years) admitted to the investigation ward for IIH stress test and who were asked to undergo two additional tests (IMGST and SST) on consecutive days. MEASUREMENTS We measured serum cortisol at baseline, 30, 45, 60, 90 and 120 minutes during the IIH stress test; baseline, 150 and 180 minutes during GST, and baseline and 30 minutes during the SST. RESULTS There was a significant rise in cortisol from baseline in all tests (P < 0.001). There was no significant difference among the peak plasma cortisol responses or the incremental rises in plasma cortisol following IMGST, SST and IIH stress test (repeated measures ANOVA F = 0.704, P = 0.503; F = 0.238, P = 0.79). The ROC plots clearly showed that the SST has poor diagnostic utility at both IIH thresholds, compared with the IMGST. CONCLUSION The peaks and incremental rises in cortisol following all three tests are comparable. Using the insulin induced hypoglycaemia stress test as a reference and peak cortisol thresholds of 500 and 580 nmol/l as discriminating variables, the short Synacthen displayed poor diagnostic utility when compared to the i.m. glucagon stimulation test. The short Synacthen may be misleading if used as a screening test as advocated by a number of authors.     

Effects of tumor-induced hyperprolactinemia on LH secretion following stimulation of the medial preoptic area, pituitary responsiveness and the estrogen-induced LH surge

In the present study we utilized the 7315a PRL- and ACTH-secreting tumor to induce a hyperprolactinemic (HP) state sufficient to profoundly suppress the postcastration LH rise in female rats. Tumor-induced prolactin levels which ranged 2,000-3,000 ng/ml substantially reduced the LH rise in both ovariectomized (OVX) and OVX + estradiol-17 beta (E2)-treated rats. Bilateral electrochemical stimulation (ECS, 100 microA DC for 60 s) of the ventral diagonal band of Broca-medial preoptic area (DBB-MPOA) resulted in comparable LH responses in control and HP rats in the presence of absence of estradiol. Transient decreases in PRL release occurred following ECS of the DBB-MPOA. Pituitary responsiveness was assessed with two LHRH challenges spaced 60 min apart at doses of 25 and 50 ng LHRH/100 g body weight. The mean maximal LH increments (delta LH) to some of these LHRH challenges were decreased in HP rats. Finally, the LH surge induced in the afternoon in OVX + E2-treated rats was diminished 71% by the presence of the PRL-secreting tumor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of dopamine on prolactin secretion and cyclic AMP metabolism in ovine pituitary cells

Prolactin secretion from cultured sheep pituitary cells was inhibited by low concentrations of dopamine (0.1 nM-0.1 microM) with a half-maximal effect at 3 nM. At a maximally effective dose (0.1 microM) dopamine significantly inhibited prolactin secretion within 5 min. with an 80% inhibition of basal secretion over 2 h. Basal prolactin secretion was stimulated by the addition of methylisobutylxanthine (MIX) (0.3-1.0 mM) and 8-bromo-cyclic AMP (2 mM), but cholera toxin (3 micrograms/ml) and prostaglandin E2 (0.1-1.0 microM), which also raised cellular cyclic AMP levels, had no effect on prolactin release. The inhibition of prolactin release by dopamine (0.1 microM) was not affected by any of these compounds. Dopamine inhibited MIX-induced cyclic AMP accumulation over a similar concentration range to the inhibition of secretion, but had no effect on the changes in cyclic AMP concentration produced by cholera toxin and prostaglandin E2. Overall the results with sheep pituitary cells suggest that lowered cyclic AMP levels do not mediate the inhibitory effects of dopamine on basal prolactin secretion, but that changes in cellular cyclic AMP levels may alter the secretion of this hormone, and dopamine may affect pituitary cell cyclic AMP concentrations in some circumstances.     

Gonadal steroid modulation of LHRH-stimulated LH secretion by pituitary cell cultures

Enzymatically dispersed rat pituitary cells were grown in primary culture, and LHRH-stimulated LH secretion was measured. Testosterone (T) decreased and 17 beta-estradiol (E) increased pituitary responsiveness to LHRH. The effect of E on LH secretion was partly due to an increase in LH content. There was a latent period of 12 h for E and 18 h for T between the onset of steroid treatment and the manifestation of steroid action. Neither steroid was required to be continuously present in order to exert its effects. After steroid withdrawal, the effect of T persisted for 72 h and that of E for more than 96 h. The actions of both steroids were blocked by protein-synthesis inhibitors. These results are consistent with the hypothesis that steroid effects rely on a mechanism involving alterations in protein synthesis; the affected proteins may be involved in the process of LHRH action.