Ubiquitination as a priming process of PKC alpha and PKC epsilon degradation in the alphaT3-1 gonadotrope cell line.

Background/aim: Protein kinase C (PKC) is a family of isoenzymes playing a key role in the regulation of gonadotrope cell functions. Specific PKC isoforms are activated and downregulated differentially by gonadotropin-releasing hormone (GnRH) and the phorbol ester TPA. In the present study, focusing mainly on PKC epsilon, the mechanisms underlying the proteasome-dependent downregulation of GnRH-activated PKC epsilon and TPA-sensitive PKC alpha and epsilon isoenzymes were investigated in alphaT3-1 gonadotrope cells.

Proteasome implication in phorbol ester- and GnRH-induced selective down-regulation of PKC (alpha, epsilon, zeta) in alpha T(3)-1 and L beta T(2) gonadotrope cell lines.

We investigated mechanisms underlying selective down-modulation of PKC isoforms (alpha, epsilon, zeta): 1) during 12-O-tetradecanoyl-phorbol-13 acetate (TPA) (10(-7) M) or GnRH (10(-7) M) desensitization conditions (2- to 6-h treatments) in two gonadotrope cell lines (alpha T(3)-1, L beta T(2)) and 2) in primary pituitary cell cultures from male rats during long-term phorbol ester administration. We demonstrated that, as in alpha T(3)-1 cells, in a more differentiated gonadotrope cell line L beta T(2) the GnRH-receptor coupling (PLC, PLA2, PLD) generated second messengers essential for PKCs activation; the characterized isoforms (alpha, beta II, delta, epsilon, zeta) were selectively and differentially down-regulated by TPA (alpha, beta II, delta, epsilon) or GnRH (delta, epsilon). In whole cell lysates, proteasome inhibitors (proteasome inhibitor I and II, Lactacystin, beta-Lactone, Calpain inhibitor I) prevented in both gonadotrope cell lines the TPA-induced depletion of PKC alpha, epsilon, and the GnRH-elicited PKC epsilon down-regulation; they counteracted in mixed pituitary cell cultures as well, the TPA-evoked PKC alpha, epsilon depletion.

GnRH signalling pathways and GnRH-induced homologous desensitization in a gonadotrope cell line (alphaT3-1).

Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively.

Luteinizing hormone-releasing hormone-signal transduction and stathmin phosphorylation in the gonadotrope alphaT3-1 cell line.

We have investigated the effects of GnRH (LHRH) and of the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate on stathmin phosphorylation in the gonadotrope alphaT3-1 cell line. Stathmin expression and its phosphorylation were maximal during the exponential phase of cell growth. LHRH stimulated stathmin phosphorylation through a specific receptor in a dose- and time-dependent manner, and TPA induced a similar extensive stathmin phosphorylation.

Differential involvement of calcium channels and protein kinase-C activity in GnRH-induced phospholipase-C, -A2 and -D activation in a gonadotrope cell line (alpha T3-1).

The mode of action of GnRH on pituitary gonadotropes involves metabolism of phospholipids, protein kinase-C (PKC) and voltage sensitive Ca2+ channels (VSCC) activation. We have studied the differential role of PKC and VSCC on the coupling of the GnRH receptor with phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities in a gonadotrope cell line (alpha T3-1), by measuring the production of inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that in these cells GnRH stimulated through a specific receptor, IPs formation, a rapid and sustained diacylglycerol generation, consequently AA release and a delayed PEt production in a dose-dependent manner.

Characterization of [hydroxyproline9]luteinizing hormone-releasing hormone and its smallest precursor forms in immortalized luteinizing hormone-releasing hormone-secreting neurons (GT1-7), and evaluation of their mode of action on pituitary cells.

[Hydroxyproline9]luteinizing hormone-releasing hormone ([Hyp9]LHRH), an endogenous hydroxylated post-translational product of the LHRH sequence, has been isolated from mammalian hypothalamus. Using the LHRH-hypothalamic cell line (GT1-7) of fetal origin, we attempted to define the substrates available for the hydroxylation process during LHRH synthesis and to characterize immunologically the [Hyp9]LHRH and pro-[Hyp9]LHRH forms with anti-LHRH antibodies of different specificities after separation by HPLC. Their biological activity and mode of action were evaluated and compared to that of LHRH and LHRH intermediate precursors in normal pituitary cells and in a gonanodotrope cell line alpha T3-1.

Gonadotropin regulation, oestrogens and the immune system.

Several transmitters and neuropeptides participate in the regulation of gonadotropins. At the hypothalamic level, control of gonadotropin releasing hormone (GnRH) involves noradrenaline, GABA, glutamate, angiotensin II, neuropeptide Y, neurotensin, and 5-hydroxytryptamine as well as interleukins 1 and 2. Ovarian steroids can interfere with gonadotropin regulation by either direct effects on GnRH release or by increasing the sensitivity of anterior pituitary cells to GnRH, thus potentiating the release of pituitary hormones.

Alpha 1-adrenergic receptor coupling with phospholipase-C is negatively regulated by protein kinase-C in primary cultures of hypothalamic neurons and glial cells.

In the present work we evaluated the interactions of adrenergic receptors with phospholipase-C (PLC) and protein kinase-C (PKC), using an in vitro system of hypothalamic neurons and astroglial cells in primary cultures. The study was performed on immature neurons after 7 days in vitro (7 Div), that is before synaptogenesis, as well as on mature cells (14 Div). Comparisons were made between neurons and glial cells at the corresponding developmental stages.

Mechanism of growth retardation following chronic administration of beta-adrenoceptor antagonists to developing rats.

A total of 112 3-week old Wistar rats were separated into eight groups: control groups I-IV (n = 62) and propranolol-treated groups V-VIII (n = 50). Propranolol hydrochloride (100 mg/kg) was present in the rats' drinking water until 26 weeks of age and growth rates of all groups were monitored daily until 53 days of age and thereafter every third day throughout the study. Chronic oral propranolol administration produced growth retardation (P less than 0.

Estradiol modulates protein kinase C activity in the rat pituitary in vivo and in vitro.

17 beta-Estradiol (E2) alters different functions of pituitary cells, including cell sensitivity to several neurohormones such as LHRH, TRH, somatostatin, or dopamine, presumably by affecting receptor coupling mechanisms. Attempting to pinpoint the membrane processes underlying this modulation, we studied the effect of E2 on pituitary kinase-C (PKC) activity, a major signal transduction enzyme. The distribution of calcium- and phospholipid-dependent partially purified PKC (chromatography on DEAE-52 cellulose columns) was evaluated in membrane and cytosol fractions from anterior pituitaries of ovariectomized (OVX) or OVX plus E2-treated rats.

Phosphorylation of a group of proteins related to the physiological, multihormonal regulations of the various cell types in the anterior pituitary gland.

We previously identified a group of cytoplasmic phosphoproteins whose phosphorylation could be related to the multihormonal regulation of PRL in the homogeneous tumor-derived GH cell lines (set of proteins 1-11) and in heterogeneous normal anterior pituitary cells in culture (set of proteins 1-15). In normal cells, a mixture of hypothalamic hormones induced, like the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, stronger phosphorylation changes than TRH alone. Proteins of the set 1-15 are therefore likely to be present also in the nonmammotrophic anterior pituitary cell types, where their phosphorylation can be regulated by the hormones specific of the cell type considered.

Differential modulation of D1 and D2 dopamine-sensitive adenylate cyclases by 17 beta-estradiol in cultured striatal neurons and anterior pituitary cells.

Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17 beta-estradiol on D1- and D2-dopamine (DA)-sensitive adenylate cyclase. 17 beta-Estradiol pretreatment (10(-9) M, 72 h) suppressed the D2-DA-induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10(-9) M, 24 h) also blocked the D2-DA-evoked response in striatal neurons whereas it enhanced by twofold the D1-DA-induced stimulation of the enzyme activity in these neurons.

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.

Variations of phospholipid methyltransferase(s) activity in the rat pituitary: estrous cycle and sex differences.

We previously demonstrated a specific stimulatory action of estrogens on phosphatidylethanolamine methylation in rat pituitary membranes. To investigate the physiological relevancy of this effect, the activity of methylating enzyme(s) was evaluated during the rat estrous cycle, a period in which both endogenous ovarian steroid levels and the sensitivity of pituitary membrane receptors fluctuate. Anterior pituitary membranes (P2) were prepared from adult female rats at different stages of the estrous cycle and assayed for phospholipid methylation in the presence of S-adenosyl-[methyl-3H]methionine as a donor of 3H-methyl groups.

Multiple coupling of neurohormone receptors with cyclic AMP and inositol phosphate production in anterior pituitary cells.

Regulation of adenohypophyseal hormone secretions has been shown to involve cyclic AMP production, modulation of phosphatidyl inositol diphosphate breakdown and Ca2+ mobilization. Various neurohormone receptors are positively or negatively coupled to adenylate cyclase activity in anterior pituitary cells. The effects of these neurohormones on adenylate cyclase activity are consistent with the effect on hormone secretions, suggesting that modulation of the enzyme activity is actually involved in the regulation of adenohypophyseal secretions.

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.

Effects of estradiol and progesterone on immunoreactive forms of hypothalamic luteinizing hormone-releasing hormone.

In order to investigate mechanisms underlying the ovarian steroid action on hypothalamic luteinizing hormone-releasing hormone (LHRH) neurons, LHRH and a higher immunoreactive molecular form (MW 1,800 daltons) of the decapeptide were immunoassayed with antibodies of different specificities in hypothalamic subcellular fractions, after molecular sieve filtration on Biogel P4 columns equilibrated with 0.2 N acetic acid containing 0.02% sodium azide.

Further evidence that thyrotropin-releasing hormone participate in the regulation of growth hormone secretion in the rat.

Effects of thyrotropin-releasing hormone (TRH) on growth hormone (GH) secretion were investigated in vivo (on intact or mediobasal hypothalamic lesioned rats tested under either anesthesia or free moving conditions) as well as in vitro (in incubation or perifusion systems of anterior pituitary tissue). The peptide induced a rapid, dose-dependent increase of plasma GH levels in free moving animals bearing an extensive lesion of the mediobasal hypothalamus including the median eminence. Under comparable conditions, TRH was ineffective in intact animals.

Progesterone-induced LHRH release in vitro is an estrogen--as well as Ca++- and calmodulin-dependent secretory process.

Mediobasal hypothalamic (MBH) slices of adult ovariectomized (OVX) rats with or without 17 beta-estradiol (E2) pretreatment, were superfused in buffered (pH 7.2) oxygenated Locke medium containing bacitracin. Pulsatile or continuous administration of progesterone (10(-7) or 10(-8) M) produced a marked increase in luteinizing hormone-releasing hormone (LHRH) release provided the animals had received E2 prior to sacrifice.

Calmodulin involvement on the Ca++-dependent release of LHRH and SRIF in vitro.

Mediobasal hypothalamic (MBH) slices of male adult rats were superfused at 37 degrees C with oxygenated Hepes-buffer Locke medium. Bacitracin (2 X 10(-5) M) was added to prevent enzymatic degradation of LHRH and SRIF. 6 min pulse of K+ (56 mM), veratridine (15 microM) or the ionophore A 23187 (10(-5) M), markedly stimulated the release of both neuropeptides.

Effects of 17 beta-estradiol on LH-RH release from rat mediobasal hypothalamic slices.

Unlabelled: Mediobasal hypothalamic slices of adult ovariectomized (OVX) rats treated or not with 17 beta-estradiol (E2) were superfused in buffered (pH 7.2) Locke medium containing bacitracin. A 6-min pulse of K+ (56 m M) was less effective in releasing luteinizing hormone releasing hormone (LH-RH) from mediobasal hypothalamic slices sampled from OVX rats than from OVX animals treated subcutaneously with either E2 or stilbestrol implants for 5 days; in contrast, the basal release of the neuropeptide was identical in both cases.

Effects of ovarian steroids on in vitro release of LHRH from mediobasal hypothalamus.

The phasic luteinizing hormone (LH) release observed in ovariectomized (OVX), estrogen-implanted rats was further amplified and advanced when progesterone (P) was given 4 h prior to the gonadotropin surge. In contrast, an inhibitory effect of P on the daily LH surge was observed when P was administered 16-36 h prior to LH peak. In order to determine whether this biphasic action of P is primarily exerted on the release of luteinizing hormone releasing hormone (LHRH), on the pituitary response to LHRH, or on both, mediobasal hypothalamic slices or pituitary fragments of adult OVX rats or of OVX rats pretreated with estrogen alone or in combination with P were tested in a perifusion system.