An inhibitor of arachidonic acid metabolism stimulates luteinizing hormone (LH) release from cultured pituitary cells

5, 8, 11, 14 eicosatetraynoic acid ("ETYA", Roche 3-1428) is a competitive inhibitor of arachidonic acid metabolism. It effectively inhibits the action of both the lipoxygenases and the fatty acid cyclooxygenases both of which utilize arachidonic acid as a substrate. In the present work, we have shown that ETYA stimulates luteinizing hormone (LH) release from cultured pituitary cells (ED50 = 10 micrograms/ml). Stimulation is not due to contaminants present in the preparation, since highly purified ETYA (characterized by GC-MS) stimulates release, while contaminants removed by silicic acid chromatography do not. In addition, neither oxidized solutions of ETYA nor arachidonic acid itself stimulate LH release. ETYA stimulated release is dose dependent and is inhibited by ions which antagonize Ca2+ action. The observation that neither indomethecin (10, 100 micrograms/ml) nor meclofenamate (1.0, 10 micrograms/ml) stimulate LH release suggests that the effect of ETYA cannot be explained by an action on cyclooxygenase. The action of ETYA may be mediated either via an effect on lipoxygenase or through some nonspecific action (such as altered membrane fluidity).     

The participation of corticosterone in luteinizing hormone releasing hormone (LH-RH) action on luteinizing hormone (LH) release from anterior pituitary cells in vitro

Corticosterone alone was not able to stimulate release of luteinizing hormone (LH) from anterior pituitary cells $\text{in}$$\text{vitro}$, but corticosterone in combination with luteinizing hormone releasing hormone (LHRH) augmented the release of LH into the culture media. These results may indicate that corticosterone may have the capacity to activate membrane receptors for LHRH in the gonadotrophs.     

Stimulatory effect of thyrotrophin-releasing hormone (TRH) on the release of luteinizing hormone (LH) from rat anterior pituitary cells in vitro

The effect of thyrotrophin-releasing hormone (TRH, 10(-7) M) on luteinizing hormone (LH) release from rat anterior pituitary cells was examined using organ and primary cell culture. The addition of TRH to the culture medium resulted in a slightly enhanced release of LH from the cultured pituitary tissues. However, the amount of LH release stimulated by TRH was not greater than that produced by luteinizing hormone-releasing hormone (LH-RH, 10(-7) M). Actinomycin D (2 X 10(-5) M) and cycloheximide (10(-4) M) had an inhibitory effect on the action of TRH on LH release. The inability of TRH to elicit gonadotrophin release from the anterior pituitary glands in vivo may partly be due to physiological inhibition of its action by other hypothalamic factor(s).     

Effects of interleukin-1 beta on secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by cultured rat anterior pituitary cells

Interleukin-1 beta (IL-1 beta) at doses of 0.15 and 1.5 nM significantly inhibited FSH secretion and stimulated LH secretion by cultured rat pituitary cells after 24-72 hr incubation whereas 15 pM of IL-1 beta was not effective. Treatment with IL-1 beta for 12-48 hr did not affect intracellular content of FSH. However, treatment with 0.15 and 1.5 nM of IL-1 beta for 72 hr significantly suppressed intracellular content of FSH whereas various doses of IL-1 beta incubated with the cells for 12-72 hr showed no effect on the intracellular content of LH. Pretreatment with IL-1 beta for 48 hr inhibited both GnRH-mediated LH and FSH secretions by the pituitary. The secretion of FSH and LH mediated by an activator of protein kinase C, phorbol 12-myristate 13-acetate, was also significantly suppressed by pretreatment with IL-1 beta for 48 hr. These results suggest that (a) IL-1 beta has opposite effects on the secretion of LH and FSH and (b) pretreatment with IL-1 beta suppresses GnRH-mediated stimulation of LH and FSH by the pituitary and this suppressive effect of IL-1 beta may involve the suppression of a protein kinase C-dependent mechanism.     

Interactions between neuropeptide Y, luteinizing hormone-releasing hormone and estradiol in the control of luteinizing hormone release from cultured ovine pituitary cells

This study used pituitary cells in culture firstly to test the hypothesis that NPY may augment the pituitary LH response to LHRH and secondly to determine whether this interaction is dependent on the presence of estradiol. LHRH (10(-10)-10(-6) M) caused a significant increase in LH secretion from dispersed ovine pituitary cells maintained in culture for six days, a response which was enhanced when cells were pretreated for three days with 4 x 10(-11) M estradiol. NPY 10(-10)-10(-6) M) had no effect on basal LH release from ovine pituitary cells maintained either in the presence or absence of estradiol. NPY (10(-10) and 10(-8) M) also had no effect on LHRH-stimulated LH release either in the presence or absence of estradiol. These results substantiate previous observations that physiologically relevant concentrations of estradiol enhance the LH response to LHRH in cultured ovine pituitary cells. However, in contrast to experiments carried out using rat pituitary cells in culture, the present data provide no evidence to support the hypothesis that NPY alone interacts with LHRH in the control of LH secretion from the ovine pituitary gland.     

Mechanism of LH release in cultured rat pituitary cells

To investigate the mechanisms of the synthesis and the release of gonadotropin, rat anterior pituitary cells were stimulated in vitro with luteinizing hormone releasing hormone (LH-RH), [D-Ser(tBu)]6 des-Gly-NH2(10) ethylamide (Buserelin) and 12-0-tetradecanoyl phorbol-13-acetate (TPA), and then the LH and LH-beta subunit released into the medium were determined by radioimmunoassay. Buserelin showed its biological activity at a much lower concentration than LH-RH, but both of them caused the release of LH and LH-beta subunit in a dose-dependent manner. Furthermore, intracellular LH synthesis from LH-beta subunit by stimulation with LH-RH or Buserelin was also found. After inducing various degrees of desensitization by stimulation with LH-RH or Buserelin in a dose-dependent manner (the first stimulation), pituitary cells were stimulated with a fixed dose of TPA (the second stimulation) and the released LH was assayed. LH was released almost constantly by the second stimulation, regardless of the dose used for the first stimulation. These results suggest that the C-kinase pathway was unaffected by the desensitization induced with LH-RH or Buserelin.     

5,6-Epoxyeicosatrienoic acid stimulates growth hormone release in rat anterior pituitary cells

The effect of arachidonic acid and some of its metabolites have been examined in rat anterior pituitary cells for their ability to release growth hormone. The cytochrome P-450 metabolite, 5,6-epoxyeicosatrienoic acid is a much more effective growth-hormone releasing agent than 15-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid methyl ester, 5-hydroxyeicosatetraenoic acid or arachidonic acid. The release of growth hormone is rapid, dose-dependent and reaches an apparent saturation after eight minutes. These studies described herein provide evidence that lipoxygenase and cyclooxygenase products of arachidonic acid are less potent while cytochrome P-450 products are more potent in the release of growth hormone from anterior pituitary cells.     

Somatostatin affects morphology and secretion of pituitary luteinizing hormone (LH) cells in male rats

The somatostatin peptides (SRIH-14, SRIH-28) and their multiple receptors are generally associated with anti-proliferative and anti-secretory actions. This study compared, using standard morphometric measurements and terminal serum LH concentrations, effects of intracerebroventricular (icv) SRIH-14 and SRIH-28 in nanomolar amounts on immunohistochemically identified LH cells in pituitary glands of male rats. Rats received l microg/5 microl of SRIH-14 or SRIH-28 icv on days 1,3, and 5, whereas control rats received only icv saline. Animals were killed 5 days later for serum LH assays. Pituitarys were harvested for PAP immunohistochemistry and morphometry. Morphometric measurements were made by an observer blinded to the treatment group. Histochemically identified LH cells from both SRIH groups appeared smaller, often pycnotic and darkly stained compared to those from saline-treated rats. Both SRIH treatments reduced (p < 0.05) the quantitative morphometric measurements for cell volume, nuclear volume, and relative volume density. Both SRIH treatments also reduced serum LH concentration (p < 0.05), supporting the hypothesis that systemic physiology was altered. Collectively, the data support the opinion that nanomolar amounts of either SRIH peptide, acting on receptors reached from cerebrospinal fluid, exert an anti-secretory effect on LH cells of male rats. Modifications of central SRIH receptors may provide an approach for treatment of male sexual dysfunction and/or be of pathophysiologic significance in these disturbances.     

Adenosine regulates the release of adrenocorticotropic hormone (ACTH) from cultured anterior pituitary cells

We have recently shown the presence of adenosine receptors coupled to adenylate cyclase in anterior pituitary and in the present studies we have investigated the effects of adenosine on ACTH release. The R-site specific analogs of adenosine such as N-Ethylcarboxamide adenosine (NECA), L-N⁶-phenylisopropyl adenosine (PIA), 2-chloro-adenosine (2-Cl-Ado) all stimulated ACTH release in a dose-dependent manner. NECA was the most potent analog and stimulated ACTH release by about 170% with an apparent Ka of 0.1 µM, whereas PIA and 2-Cl-Ado were less potent and stimulated the release by about 110% and 125% with an apparent Ka of 0.2 and 0.4 µ-M respectively. The stimulation of ACTH release by NECA was inhibited by 3-isobutyl-1-methylxanthine (IBMX). On the other hand, adenosine deaminase (ADA) treatment of the cells also stimulated ACTH release as well as adenylate cyclase activity by about 2-fold, suggesting that endogenous adenosine plays an inhibitory role in the release of ACTH. Other agents, such as corticotropin-releasing factor (CRF), vasoactive intestinal peptide (VIP) and forskolin (FSK) also stimulated ACTH release from these cells. In addition, the stimulation by an optimal concentration of NECA was almost additive with maximal stimulation caused by VIP and FSK. These data suggest that adenosine modulates ACTH release from anterior pituitary through its interaction with adenosine receptors coupled to adenylate cyclase.Abbreviations NECA N-Ethylcarboxamideadenosine - PIA L-N⁶-Phenylisopropyladenosine - 2-Cl-Ado 2-chloroadenosine - FSK Forskolin - VIP Vasoactive Intestinal Peptide - CRF Corticotropin Releasing Factor - ADA Adenosine Deaminase - IBMX 3-Isobutyl-1-methylxanthine     

Endogenous arachidonic acid metabolism by cultured arterial smooth muscle cells

Cultured aortic smooth muscle cells originated from healthy and atherosclerotic rabbits produce prostaglandins (namely prostacyclin) at a basal state. Prostaglandin secretion is dramatically reduced in atherosclerotic cells. This impairment was not correlated with any alteration of acyl hydrolase activities and probably involved a decrease of cyclooxygenase activities.     

Insulin-like growth factor I enhances gonadotropin-releasing hormone-stimulated luteinizing hormone release from bovine anterior pituitary cells

The role of insulin-like growth factor I (IGF-I) in the release of luteinizing hormone (LH) is unclear in ruminants. In the present study, the effects of IGF-I on the release of LH stimulated by gonadotropin-releasing hormone (GnRH) were examined in primary cultures of bovine anterior pituitary (AP) cells, and the interaction between estradiol-17beta (E(2)) and IGF-I was characterized. GnRH(100nM)-stimulated LH release from the cultured cells was increased (P<0.05) 12, 24 and 36h after addition of IGF-I (250ng/ml), with a maximum at 12h (48.4ng/ml media versus 35.4ng/ml media in controls). IGF-I at concentrations of 25, 250 and 500ng/ml increased the release by 18.7, 24.2 and 28.9%, respectively (P<0.05), when compared with controls (37.2ng/ml media). E(2) (10nM), IGF-I (250ng/ml) and combined treatment of E(2) plus IGF-I also induced significant increases in LH release (P<0.05). The amounts of LH release after treatment with E(2) alone was 37.3% greater than with IGF-I alone (39.0ng/ml media versus 28.4ng/ml media) (P<0.05). When E(2) and IGF-I were added together (45.6ng/ml media), the release of LH was significantly greater than with either E(2) alone or IGF-I alone (P<0.05). E(2) (10nM) significantly (P<0.05) increased the amount of GnRH bound to the cells by 51.6% when compared with controls, however, IGF-I (250ng/ml) failed to increase GnRH binding. These results show that IGF-I enhances GnRH-stimulated LH release without changing the number of GnRH receptors in cattle, and IGF-I interacts with E(2) to increase the response to GnRH.     

Vasoactive intestinal peptide stimulates luteinizing hormone-releasing hormone release from median eminence synaptosomes

Third ventricular injections of vasoactive intestinal polypeptide (VIP) result in increased circulating levels of luteinizing hormone (LH) in conscious, freely moving, ovariectomized (OVX) rats. This effect of VIP has been hypothesized to be mediated via stimulation of luteinizing hormone-releasing hormone (LH-RH) secretion from hypothalamic neurons since VIP is incapable of stimulating LH release from rat pituitaries in vitro. To test this hypothesis, crude synaptosomes were prepared from OVX rat median eminence (ME) tissue. Release of LH-RH from these preparations displayed time and temperature dependencies. Additionally, depolarization-induced (elevated K⁺) LH-RH release was demonstrated to be Ca²⁺-dependent. VIP, in doses ranging from 1.5 · 10^?9 M, was capable of stimulating significantly greater LH-RH release from ME synaptosomes than that from control preparations. VIP's close structural homolog, glucagon, was incapable at the same doses of stimulating increased LH-RH release. These findings offer an explanation for the effect of third ventricularly injected VIP on LH release and suggest a modulatory role for VIP in the hypothalamic control of LH secretion.     

Lactacystin stimulates arachidonic acid metabolism in rat liver cells: effects of cell density on arachidonic acid release, PGI2 production and cyclooxygenase activity

Lactacystin, an inhibitor of proteasome activity, amplifies prostaglandin I2 production by rat liver cells stimulated by 12-O-tetradecanoylphorbol-13-acetate, transforming growth factor-alpha or interleukin-1. Lactacystin also stimulates the cell's release of arachidonic acid (AA) and increases the cyclooxygenase activity in these cells. In serum deprived cells, the enhanced AA release is reduced, cyclooxygenase activity on exogenous AA is increased and endogenous production of prostaglandin I2 is unchanged. These findings suggest that, in vivo, the ratio of dividing to quiescent cells in a tissue may influence eicosanoid production. The increases in prostaglandin I2 production, AA release and cyclooxygenase activity on exogenous AA resulting from the combined lactacystin and 12-O-tetradecanoylphorbol-13-acetate treatment are inhibited by actinomycin or cycloheximide.     

Albumin stimulates the release of arachidonic acid from phosphatidylcholine in hamster lungs

¹⁴C-Arachidonic acid injected into the pulmonary circulation of isolated hamster lungs was effectively incorporated into lung lipids. Once retained the radiolabel was relatively stable but the release of radioactivity increased up to 10-fold when bovine serum albumin (1 %) was added to the perfusate. This efflux of radioactivity was not blocked by quinacrine, a phospholipase A₂ inhibitor. In albumin experiments the released ¹⁴C-araehidonate griginated mainly from the phospholipid fraction in which phosphatidylcholine was the main source of the released radioactivity.Pulmonary infusion of albumin had no significant effect on the amount of ¹⁴C-arachidonic acid in the neutral lipid or free fatty acid fractions of perfused lungs. In experiments with albumin about 80 % of the released radioactivity co-chromatographed with unlabelled arachidonic acid whereas in the absence of albumin only about 20 % of the released radioactivity was unmetabolized arachidonic acid. This study indicates that albumin stimulates the release of arachidonic acid from isolated hamster lungs and that the release is increased mainly from the phosphatidyl choline fraction.