Zvi Naor

Role of Dynamin, Src, and Ras in the Protein Kinase C-mediated Activation of ERK by Gonadotropin-releasing Hormone

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gβγ, and β-arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.

Acute Effect of ▵1–Tetrahydrocannabinol on the Hypothalamo-Pituitary-Ovarian Axis in the Rat

Administration of delta1-tetrahydrocannabinol (delta1-THC), the principal psychoactive ingredient of cannabis, to proestrous rats (2 mg/rat, i.p., between 12.00 and 16.00 h) suppressed the proestrous rise in the plasma levels of LH, FSH and prolactin (Prl) and caused a 24 h delay in ovulation. Furthermore, the increased accumulation of prostaglandins of the E-type (PGE) in the ovaries, normally seen on the evening of proestrus, was prevented. Earlier (08.00-10.30 h) or later (18.00 h) administration of the drug on the day of proestrus was only partially effective in inhibiting ovulation. The suppressive effects of delta1-THC on ovulation and gonadotropin secretion were prevented by administration of gonadetropin releasing hormone (GnRH, 0.2 microgram/rat) 1 h after the drug, indicating that the central action of delta1-THC was exerted on the hypothalamus and not on the pituitary gland. Administration of ovine luteinizing hormore (oLH, 2.5 microgram/rat at 16.30 h on the day of proestrus restored ovulation and ovarian PGE accumulation in Nembutal-treated rats, but not in delta1-THC-treated rats; higher doses of oLH (5-10 microgram/rat) reversed the action of delta1-THC on these two parameters.

c-Src Is Activated by the Epidermal Growth Factor Receptor in a Pathway That Mediates JNK and ERK Activation by Gonadotropin-releasing Hormone in COS7 Cells

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated Gβγ and β-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.

Mechanism of action of gonadotropin releasing hormone: Role of lipoxygenase products of arachidonic acid in luteinizing hormone release

The mechanism of action of gonadotropin-releasing hormone (GnRH) upon pituitary luteinizing hormone (LH) secretion has not yet been elucidated, but recent evidence has suggested that arachidonic acid or its metabolites are involved in GnRH action. In cultured rat pituitary cells, arachidonic acid and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) elicited concentration-dependent release of LH with EC50 of about 12 microM. Other lipoxygenase derivatives including 11-, 12- and 15-HETE, had no consistent effect on LH release, and leukotrienes (B4 and C4) exerted only minor stimulatory actions on LH release. The lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA), 5,8,11,14-eicosatetraynoic acid (ETYA), and 3-amino-1-(3-trifluoromethyl phenyl)-2-pyrazoline hydrochloride (BW 755C) caused dose-dependent inhibition of GnRH-induced LH release, with IC50 values of 5, 8.5, and 175 microM, respectively. In contrast, the cyclooxygenase inhibitor, indomethacin, had a biphasic action on GnRH-stimulated LH release, with potentiation of GnRH action at low doses (up to 25 microM) and no effect at higher concentrations. These findings are consistent with the potential role of a 5-lipoxygenase product of arachidonic acid in the mechanism of action of GnRH on pituitary gonadotropin release.

Possible involvement of lipoxygenase products of arachidonic acid pathway in ovulation

The possible involvement of products of the lipoxygenase pathway of arachidonic acid cascade in ovulation was tested by intrabursal injection of nordihydroguaiaretic acid (NDGA); 5, 8, 11-eicosatriynoic acid (5, 8, 11-ETYA), 3 amino-1-(3 trifluromethyphenyl)-2-pyrazoline hydrochloride (BW755c) and (FPL 55712). All these drugs reduced the number of ova released from the treated ovaries in a dose-dependent manner, without affecting ovulation from contralateral ovaries. NDGA was most potent since it completely blocked ovulation from the treated ovaries in 17/38 rats receiving a dose higher than 0.15 mg/bursa. This effect of NDGA cannot be ascribed to its inhibition of ovarian PGE synthesis. Conversion of labeled arachidonic acid via the lipoxygenase pathway by preovulatory rat follicles was demonstrated by TLC chromatography. Collectively, these results suggest the involvement of products of lipoxygenase pathway of arachidonic acid in ovulation in the rat.

Compartmentalization of luteinizing hormone pools: Dynamics of gonadotropin releasing hormone action in superfused pituitary cells

Cultured rat anterior pituitary cells were continuously perfused with medium 199, and exposed to short (5 min), intermediate (30 min), or long (6 h) pulses of a maximally effective concentration of gonadotropin-releasing hormone (GnRH). Assay of the effluent by radioimmunoassay and interstitial-cell bioassay revealed a biphasic response to GnRH, and indicated that 3 pools of luteinizing hormone (LH) are present in the gonadotroph. A rapidly releasable peak of bioactive LH comprising about 2% of the total cellular LH was mobilized within 1 min of GnRH addition, lasted for 3-4 min, and was independent of the duration of stimulation. The second, larger pool of bioactive LH varied from 15 to 50% of the total LH as the duration of exposure to GnRH was increased from 5 min to 6 h. A third LH pool comprising up to 50% of the total LH could be mobilized by 50 mM potassium but not by continuous GnRH treatment, due to refractoriness of the cells to prolonged stimulation by the decapeptide. In contrast, repeated pulses of GnRH evoked a series of biphasic LH peaks with profiles similar to that observed during a single response to GnRH, indicating that continuous exposure to GnRH is necessary for densensitization. Release of LH from the perfused cells was calcium-dependent, and the bio-immuno ratio of the first and second pools of LH was similar. The in vitro secretion profile of cultured rat cells is comparable with the early and late phases of LH release observed in GnRH-infused man, but occurs much more rapidly, and demonstrates heterogeneity of the LH release process at the level of the gonadotroph. The superfusion technique provides a powerful tool to further investigate the bioactivity of GnRH and its analogs for use in fertility control.

Pituitary cyclic AMP production and mechanism of luteinizing hormone release

Secretion of anterior pituitary luteinizing hormone (LH) is controlled by the hypothalamic luteinizing hormone-releasing hormone (LH-RH) which has recently been isolated [ 1,2] . Crude hypothalamic extract containing LH-releasing activity was reported to stimulate adenylate cyclase and to increase the cyclic AMP level in the pituitary gland [3] . Recently it was found that synthetic LH-RH increased adenylate cyclase activity [4] cyclic AMP level and concomitantly stimulated LH release [S-8] . Hence it was suggested that cyclic AMP is the mediator of LH-RH action on LH release [S-7]. This conclusion was further supported by the findings that analogues of LH-RH which increased cyclic AMP production also caused LH release [9] . The addition of dibutyryl cyclic AMP (DBC) to the incubation medium was reported to enhance the release of LH [7,10] , while other workers found no increase of LH upon the addition of this drug [ 11,121 . The present investigation was intended to clarify whether the increase in pituitary cyclic AMP production by LH-RH is an obligatory step in LH release.

Stimulatory effect of prostaglandin E2 on LH release in the rat: evidence for hypothalamic site of action.

The intravenous administration of prostaglandin E2 (PGE2; 100 mug/rat) to immature male and female rats increased the serum level of lutenizing hormone (LH). This effect was prevented by the prior administration of an antiserum to the hypothalamic gonadotropin-releasing hormone. LH release from rat pituitaries in vitro was not stimulated by PGE2 (10 mug/ml). It is inferred that the stimulatory effect of PGE2, on LH release is not a direct one on the pituitary gland, but is exerted at the level of the hypothalamus.

Cytochemical characterization of pituitary target cells for biotinylated gonadotropin releasing hormone

These studies describe the application of new cytochemical stains that co-localize a biotin-labeled gonadotropin releasing hormone (GnRH) analog and FSH or LH in the same field or cell. Pituitary monolayer cells were stimulated with the [D-Lys6] GnRH analog or the same analog labeled with biotin. Biotinylated [D-Lys6] GnRH exhibited a higher affinity and was 7-10 X more potent than unlabeled [D-Lys6] GnRH. The avidin-biotin peroxidase complex technique (ABC) was applied to localize the biotinylated GnRH on the cells with the use of a dense black peroxidase substrate. Specificity tests showed that the stain could be eliminated by competition with unlabeled [D-Lys6] GnRH. The GnRH stain was followed by immunocytochemical stains for LH beta, FSH beta or 25-39ACTH with a different peroxidase substrate (amber or orange-red). Stain for GnRH was found on the surfaces of 16% of the cells and 60-90% of the GnRH stained cells also stained for one of the gonadotropins. Most (90-100%) of the gonadotropes showed stain for GnRH. Our studies demonstrate that a potent biotinylated GnRH analog binds cells that can be identified specifically as gonadotropes.

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied in vitro by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10(-7) M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [125I]iodo-[D-Ala6]des-Gly10-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.