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We investigated the kinetics of gonadotropin-releasing hormone (GnRH)-induced activation of the protein kinase C (PKC) δ isoform in αT3-1 gonadotrope cells. Results were evaluated in subcellular fractions and whole-cell lysates using specific antibodies recognizing either non- or (trans- and auto-)phosphorylated forms of the kinase at Thr505 and Ser643 residues modulating stability and/or activation of the enzyme. Under basal conditions, and in contrast to PKC Ε, PKC δ was mainly associated with the membrane compartment. GnRH (10–7M) elicited further and rapid membrane translocation and time-dependent phosphorylation at both sites of PKC δ. The neuropeptide’s effects did not show a refractory period after short but successive GnRH stimulation and were abolished by the GnRH antagonist, antide. Sustained GnRH stimulation (2–6 h) provoked rapid down-regulation of PKC δ. Antide, by inhibiting the initial processes (translocation, phosphorylation), counteracted the degradation of the enzyme. Proteolytic processing of PKC δ was shown to mainly involve proteasome activity. Indeed, specific proteasome inhibitors prevented GnRH-elicited kinase depletion and induced membrane accumulation of the enzyme in a phosphorylated (Thr505, Ser643) form. Thus, GnRH may regulate time-dependent cell responses by modulating the phosphorylation/activation state of its signal transduction effector proteins, and by maintaining their appropriate expression balance via proteolytic processes involving the proteasome system.

We investigated mechanisms underlying selective down-modulation of PKC isoforms (α, e, ζ): 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 (αT3-1, LβT2) and 2) in primary pituitary cell cultures from male rats during long-term phorbol ester administration. We demonstrated that, as in αT3-1 cells, in a more differentiated gonadotrope cell line LβT2 the GnRH-receptor coupling (PLC, PLA2, PLD) generated second messengers essential for PKCs activation; the characterized isoforms (α, βII, δ, e, ζ) were selectively and differentially down-regulated by TPA (α, βII, δ, e) or GnRH (δ, e). In whole cell lysates, proteasome inhibitors (proteasome inhibitor I and II, Lactacystin, β-Lactone, Calpain inhibitor I) prevented in both gonadotrope cell lines the TPA-induced depletion of PKC α, e, and the GnRH-elicited PKC e down-regulation; they counteracted in mixed pituitary cell cultures as well, the TPA-evoked...

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 (αT3-1) 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. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. αT3-1 cells possess several PKC isoforms which, except PKCζ, were differentially down-regulated by TPA (PKCα, βII, δ, e, η) or GnRH (PKCβII, δ, e, η). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on GnRH-induced IPs, AA and PEt formation remained unchanged. In conclusion, in αT3-1 cells the GnRH-induced homologous desensitization affects the GnRH coupling with PLC, PLA2 and PLD by mechanism(s) which do not implicate TPA-sensitive PKC isoforms, but likely reflect time-dependent modification(s) on the activation processes of the enzymes.

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. In contrast to GnRH-induced PLC activity, the PLA2 and PLD stimulation by the neuropeptide involved Ca2+ mobilization via VSCC activation. BAY-K8644 a VSCC agonist significantly potentiated, while the VSCC antagonist nitrendipine markedly inhibited GnRH-induced AA release and PEt production. TPA, a phorbol ester which induced a rapid and important redistribution of PKC, although unable to elicit PLC or PLA2 stimulation, specifically provoked PLD activation in a PKC-dependent but Ca(2+)-independent manner. The PKC stimulation by TPA significantly inhibited the GnRH-stimulated IPs and AA formation, while it potentiated the GnRH-evoked PEt production. This negative feed-back of PKC on GnRH-Induced PLC and PLA2 activities was reversed when PKC was either down regulated after long TPA treatments or inhibited by the PKC inhibitors, staurosporine or GF109203X. The GnRH-induced PEt formation was markedly diminished in PKC depleted cells or after PKC inhibition. Under such conditions, both agonist and antagonist of VSCC became less effective in modulating the remaining GnRH-evoked PEt formation. These results suggest that PKC, in coordination with Ca2+, plays a key role in regulating the cross-talk between the multiple phospholipases implicated in the GnRH signal transduction.

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. Norepinephrine (NE) increased inositol phosphates (IPs) formation in a dose- and time-dependent manner. The NE effect was mediated by alpha 1-receptor (alpha 1R) and was observed in young cells before synaptogenesis as well as in mature neuronal cultures; its amplitude was enhanced during the latter stage of the neuronal development. The coupling of alpha 1R with PLC was partially sensitive to pertussis toxin treatment and did not implicate the activation of calcium voltage-dependent channels. Activation of PKC by 12-O-Tetradecanoylphorbol 13-acetate (TPA) inhibited in a time-dependent manner the NE-stimulated production of IPs in young and mature hypothalamic neurons; however, in PKC depleted cells NE-induced IPs formation remained unchanged. In hypothalamic astroglial cell cultures the adrenergic stimulus of IPs generation was also mediated by alpha 1R. The effect was observed at both developmental stages, with a greater response in 14 Div cultures, and was insensitive to pertussis toxin treatment. As in neurons, activation of PKC resulted in inhibition of NE-induced IPs formation. These data indicate that functional interrelation between alpha 1R, PLC, and PKC is already present in immature neurons and glial cells and progressively develops in culture.

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.In pull-down assays involving the use of glutathione-agarose affinity beads conjugated with a GST-fusion protein containing ubiquitin-associated domains of Rad23 that bind very likely to K48-linked polyubiquitinated proteins, TPA induced rapid (within 15 min) and sustained (up to 4 h) PKC alpha and PKC epsilon polyubiquitination. However, GnRH selectively elicited receptor-dependent polyubiquitination of PKC epsilon, but not that of PKC alpha. The GnRH-evoked PKC epsilon polyubiquitination was a strong, fast process (taking place as early as 10 min) which decreased progressively with time (but was still detectable after 4 h of treatment). In addition, no apparent association between PKC epsilon and the lysosomal compartment was observed upon performing double-labeling immunofluorescence and confocal microscopy, after either 10 min or 1 hour of stimulation by GnRH or the phorbol ester.In alphaT3-1 gonadotrope cells, polyubiquitination is therefore the event triggering GnRH-evoked PKC epsilon desensitization as well as TPA-induced PKC alpha and PKC epsilon downregulations; it precedes the respective isoenzyme's degradation by the proteasome complex.

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. In contrast, the inhibitors of calpain(s) and lysosomal proteases (Calpeptin, E64d, Calpain inhibitor II, and PD150606), were ineffective. As shown in alpha T(3)-1 subcellular fractions, proteasome abrogation did not affect membrane translocation of TPA- and GnRH- target isoforms (alpha, epsilon) but, preventing their degradation, favored enzyme accumulation to the membrane compartment. Proteolysis processing of PKCs may be dependent upon their phosphorylated state and/or catalytic activity. Inhibition of PKC catalytic activity (GF109203X, Gö6976), selectively prevented the TPA-evoked PKC alpha depletion in both mixed pituitary cells and alpha T(3)-1 gonadotropes; in alpha T(3)-1 subcellular fractions, PKC alpha inactivation overcame the TPA-evoked isoenzyme degradation by inducing a pronounced membrane accumulation of the isoform without affecting its membrane relocalization. Thus, the proteasome system by adjusting PKC cellular levels, may represent a regulatory proteolytic pathway implicated in the adaptive mechanisms of the time dependent cell responses.

[Hydroxyproline 9 ]luteinizing hormone-releasing hormone ([Hyp 9 ]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 [Hyp 9 ]LHRH and pro-[Hyp 9 ]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 αT3-1. LHRH-like immunoreactivity was progressively increased in cells and media during cell culture. [Hyp 9 ]LHRH and its two smallest precursor forms ([Hyp 9 ]LHRH-(Gly 11 ) and -(11–13)) were detected in cells and in media. They were simultaneously detected with the homologous LHRH molecular forms indicating that the hydroxylation occurs early in the processing of pro-LHRH. [Hyp 9 ]LHRH-like molecules were more abundant than LHRH forms in media. This predominant release may thus represent a physiological process occurring during fetal life. Free acid forms of both decapeptides were detected only in cells. Furthermore, the results obtained suggest that conversion of Gln 1 in pyroGlu 1 occurs before or during processing into the hydroxylated or non-hydroxylated LHRH intermediate (11–13)-precursors. The biosynthetic pathway is thus common for both decapeptides and it is not altered by the hydroxylation process. LHRH and [Hyp 9 ]LHRH shared the same receptor for their biological activity, as assessed by measuring luteinizing hormone release and activation of phospholipase C and A 2 . [Hyp 9 ]LHRH was, however, less potent than LHRH.

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. E2 administration by implants to OVX animals increased significantly both soluble and particulate enzyme activity. The effect increased progressively from 24 h to 5 days after E2 treatment. Administration of 17 alpha-estradiol, an inactive stereoisomer of E2, was ineffective, pointing to stereospecific interaction. Total destruction of neural connections to the pituitary (complete hypothalamic lesions) did not modify the enzyme response to E2 administration, indicating a direct effect of the steroid on pituitary PKC activity. A direct E2 (10(-9) M) effect was confirmed in primary mixed cultures of pituitary cells; it was time dependent (15-96 h) and specific, and reflects a genomic E2 action. E2 treatment for shorter times had no effect on the enzyme levels or the membrane redistribution of PKC activity. In contrast, under the same experimental conditions phorbol esters (12-O-tertadecanoyl-phorbol-13-acetate (TPA] induced a rapid and sustained translocation of the enzyme. PKC activity was found in all pituitary cell types, with maximal activity in fractions of gonadotropes and thyrotropes, as evaluated in cultures enriched in certain types of pituitary cells separated by means of unit gravity gradient sedimentation. E2 treatment (10(-9) M; 72 h) significantly increased both soluble and particulate enzyme levels in all cell types. In addition, administration of E2 (10(-9) M; 72 h) to cell cultures strongly increased the TPA-evoked LH and PRL release. These results indicate that E2-induced changes in pituitary function include selective effects of the steroid on PKC activity involved at different levels in the coupling mechanisms.

This study was intended to examine the role of hypothalamic norepinephrine (HNE) and dopamine (HDA) in episodic luteinizing hormone (LH) release in adult ovariectomized rats. Unrestrained, unanesthetized rats with indwelling right atrial cannulae were bled continuously (30, 50, or 100 mul of whole blood/4-6 min for 3-4 hours), and the blood samples were analyzed for LH by radio-immunoassay. In other individual rats, changes in the hypothalamic levels of norepinephrine and dopamine after drug administration were determined by a radioisotopic-enzymatic catechol-O-methyl transferase assay. alpha-Methyl-p-tyrosine significantly decreased HNE and HDA concentrations but failed to alter episodic LH release. Two dopamine-beta-hydroxylase inhibitors (U-14,624 and FLA-63) caused marked reductions in HNE, small but not statistically significant increases in HDA, and an inhibition of episodic LH secretion. Apomorphine, a dopamine receptor stimulator, caused a transient (50-60 min) but marked inhibition of episodic LH release. Saline injection had no effect. Pimozide, a blocker of dopamine receptors, prevented the inhibitory effects seen following apomorphine. Although not studied in detail, pimozide alone did not appear to alter episodic LH secretion. These data suggest that in adult ovariectomized rats norepinephrine may be an excitatory neurotransmitter in the modulation of episodic LH release. The activation of dopamine receptors may be capable of inhibiting this release process. However, the apparent inability of pimozide alone to alter episodic LH discharge suggests that under physiological conditions dopamine may not play a role in the modulation of episodic LH secretion.

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.

The circadian pattern of LH release observed in ovariectomized estrogen-implanted rats was inhibited by blockade of alpha-adrenergic receptors by phenoxybenzamine; in contrast, blockade of beta-receptors by propranolol was ineffective. Prasozin, an alpha 1-antagonist, was as effective as phenoxybenzamine, whereas yohimbine, an alpha 2-antagonist, was effective only at high doses. Neither phenoxybenzamine nor prazosin were able to modify the LHRH-induced release of LH from superfused pituitaries. These results suggest an involvement of hypothalamic alpha 1-receptors in the control of circadian LH release.

A superfusion system was used in order to investigate the ionic requirements of luteinizing hormone releasing hormone (LHRH) and somatostatin (SRIF) release from mediobasal hypothalamic (MBH) slices of male adult rats. Slices were superfused with Hepes-buffered Locke medium at 37 °C in an atmosphere of Ch 95%–CO2 5% for 1 h. Bacitracin (2 × 10–5M) was added to the medium to prevent enzymatic degradation of neuropeptides. Depolarizing agents such as potassium (K+) or veratridine stimulated LHRH and SRIF release in a dose-dependent manner. Maximal effect was obtained with K+ 56 mM and veratridine 50 µM. The depolarizing effect of K+ 56 mM was specific and not due to the hypertonicity of the medium used. Neither Mg2+ nor chlorine was needed for the spontaneous or K+-evoked release of LHRH and SRIF. The amplitude of the secretory response to K+ 56 mM was related to Ca2+ concentration tested in the range of 0.2–8.8 mM; maximal responses were obtained between 0.8 and 1.8 mM. Removal of Ca2+ from the medium with or without replacement by Mg2+, as well as administration of voltage-sensitive Ca2+ channel blockers (D-600 10–4M, Mn2+ 3 mM) blocked both K+ and veratridine induced neuropeptide release. When sodium channels and the ‘early’ calcium channels were blocked by tetrodotoxin (5 × 10–7M) the stimulatory effect of veratridine was completely blocked whereas the stimulation of K+ was unaffected. These experiments indicate that: (1) both K+ and veratridine induced-LHRH and SRIF release is a Ca2+-dependent process; (2) Ca2+ concentration is critical for the amplitude of the secretory response; (3) the main Ca2+ channel involved in neuropeptide release corresponds to the voltage-sensitive Ca2+ channel, and (4) neither magnesium or chlorine is needed for either spontaneous or evoked release of LHRH and SRIF.

Stimulation of dopamine receptors by apomorphine inhibits episodic LH release in ovariectomized rats. The present study was designed to examine further the role of dopamine in this process. Unrestrained, unanesthetized rats with indwelling right atrial cannulae were bled continuously (30 or 50 microliters of whole blood/5 min for 3-6 h) and whole blood samples analyzed for LH by radioimmunoassay. Animals were treated with various compounds reported to stimulate or block dopamine receptors. ET 495, a long acting dopamine receptor stimulating agent, caused a marked inhibition of episodic LH release (2 1/2-4 h). Control injections of distilled water had no effect. d-Butaclamol, a blocker of dopamine receptors, did not itself alter episodic LH release but prevented the inhibitory effects seen following apomorphine or ET 495. I-butaclamol, a biologically inactive form of butaclamol, had no effect. Measurement of plasma corticosterone levels in these same animals indicated increased values following apomorphine or ET 495 alone (when LH release was inhibited), as well as after apomorphine or ET 495 administration to d-butaclamol-pretreated rats (when LH levels did not change). These data support our previous hypothesis that in ovariectomized adult rats, activation of dopamine receptors is capable of inhibiting episodic LH release, but that dopamine may not play an inhibitory role under normal physiological conditions in the modulation of LH secretion. In addition, the inhibitory action of apomorphine and ET 495 does not appear to be exerted via a stress-induced release of adrenal corticosterone.

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. After chloral hydrate anesthesia a GH response to TRH was recorded in both groups, but lesioned rats exhibited a better responsiveness to all doses tested. In vitro TRH increased GH release from incubated or perifused pituitaries sampled from both intact and lesioned rats in a transient and concentration-dependent manner. A similar effect was obtained with the (3 Me His2) analogue of TRH. These findings indicate that TRH can affect GH secretion at the pituitary level under specific experimental conditions and support the hypothesis that either peripheral hormones or other, still unidentified hypothalamic neurohormones may modulate this effect.

Mediobasal hypothalamic (MBH) slices of male adult rats were superfused at 37 °C with oxygenated Hepes-buffer Locke medium. Bacitracin (2 × 10–5MJ was added to prevent enzymatic degradation of LHRH and SRIF. 6 min pulse of K+ (56 mM), veratridine (15 µM) or the ionophore A 23187 (10–5M), markedly stimulated the release of both neuropeptides. Trifluoperazine, a calmodulin inhibitor, decreased the K+-evoked LHRH and SRIF release in a dose-dependent manner; it was also effective in inhibiting the veratridine-induced neuropeptides release. Phenytoin, a calmodulin-dependent kinase inhibitor, also decreased in a dose-dependent manner the K+-induced LHRH and SRIF release; the basal release of both neuropeptides remained unaffected by either treatment. The ionophore-stimulated release of both neuropeptides was significantly inhibited as well. These data demonstrate that a Ca++-calmodulin kinase system may be involved in the mechanism of depolarization-induced LHRH and SRIF release from hypothalamic nerve terminals.

Mediobasal hypothalamic (MBH) slices of adult ovariectomized (OVX) rats with or without 17β-estradiol (E2) pretreatment, were superfused in buffered (pH 7.2) oxygenated Locke medium contain

Opiate binding sites as well as LHRH and SRIF content were evaluated in mediobasal hypothalamus (MBH) from normal male rats and animals subjected to anterolateral deafferentation of the hypothalamus. A 87 and 44% depletion of LHRH and SRIF content respectively and a 50% decrease in the number of specific opiate binding sites was observed in the deafferented MBH. Dopa-de-carboxylase activity remained unchanged. These data indicate that presynaptic opiate binding sites are present on LHRH and SRIF mediobasal hypothalamic nerve endings.

Publisher Summary This chapter discusses brain amines and hypothalamic control of pituitary gonadotropic secretion. Brain neurotransmitters are mainly involved in gonadotropic modulation under conditions of hormone fluctuations. This is the case during circadian, cyclic, or pulsatile stimulation by the hypothalamus of hormone release. Pulsatile secretion is of particular importance whenever high levels of luteinizing hormone (LH) are secreted—for instance, after castration in rats during the mid-cycle LH peak observed in primates. Noradrenergic inputs to the preoptic area of the hypothalamus facilitate this mode of secretion, mainly via α-adrenergic receptors, whereas dopamine neurons antagonize it. This is an effect that likely accounts for the inhibitory effect of the amine on LH plasma levels observed after castration. Mesencephalic serotonin neurons, projecting to the suprachiasmatic nucleus of the hypothalamus, play a major role in controlling circadian hormone secretion; this account for the effect of drugs affecting 5-HT metabolism or receptors on the strictly timed ovulation processes of rodents. Dopamine and serotonin fibers, innervating the median eminence, can also affect directly LHRH release from neurosecretory nerve endings, respectively, by facilitating or inhibiting it.

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.

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. Mediobasal hypothalamic slices were perifused in buffered (pH 7.2) oxygenated Locke's medium containing bacitracin (2 X 10(-5) M). In the absence of estrogen pretreatment, high (56 mM) concentrations of K+ were barely effective in releasing LHRH. Subcutaneous implantation of 17 beta-estradiol for 5 days markedly increased the amplitude of the LHRH secretory response to K+ depolarization. Additional administration of P (25 mg/rat s.c.) 4 h before sacrifice further amplified the K+-induced LHRH release. In contrast, the K+-evoked LHRH secretion was significantly inhibited when P was given 16 or 36 h before. Estradiol thus appears to facilitate the LHRH secretory response to depolarizing stimuli, whereas P either enhances or blocks the induced LHRH release depending upon its time of administration. At the pituitary level, the sensitivity of LHRH-induced LH release was also increased after estrogen pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)

This study examined alterations in episodic LH release in response to prolonged, slow infusions of dopamine (DA), norepinephrine (NE), or epinephrine (EPIN) into the third ventricle in adult, ovariectomized (OVX) rats, and the influence of priming with ovarian steroids on the LH response to the catecholamines. Unanesthetized rats with right atrial cannulae were bled continuously at slow rates for 1–1½ h prior to infusion, 1–1½ h during infusion, and up to 1 h afterwards. The amines were protected from oxidation by ascorbic acid, and infused in an artificial cerebrospinal fluid (CSF) vehicle (pH 7.29-7.33) into the third ventricle at a rate of 25–27 μl/h. Blood samples were analyzed for LH by radioimmunoassay. In unprimed, OVX rats, infusions of artificial CSF, as well as low doses of DA (2–4 μg/h) or NE (0.3–0.6 μg/h), had no effect on episodic LH release or mean blood LH levels. However, administration of 8.5 and l7μg DA/h, and 5.5 and 11 μg NE/h, resulted in a decrease in blood LH levels and, in most animals, prolonged intervals between peak blood LH levels during infusion or inhibitions which began rapidly and lasted for nearly the entire infusion period or longer. In contrast, infusion of 5.7 and 11.5 μg EPIN/h had no effect on blood LH levels in unprimed rats. In OVX rats primed 3 days prior to infusion with 50 μg estradiol benzoate and 25 mg progesterone (OEP), administration of CSF or the same doses of DA that previously inhibited episodic LH release had no effect on LH secretion. However, these steroids completely reversed the LH response to 11 μg NE/h, with increases in LH release occurring during infusion. EPIN, in doses ineffective in unprimed rats (5.7 and 11.5 μg/h), also caused elevations in blood LH levels in OEP rats. Finally, in rats primed with 5 μg estradiol benzoate/100 g b.w./day for the 2 days prior to infusion, none of the three neurotransmitters had any effect on LH release. These experiments indicate that in the absence of ovarian steroids intraventricular administration of DA can decrease mean blood LH levels and episodic LH release, and that this amine has no excitatory effect on LH release in steroid-primed rats. In contrast, NE and EPIN increased LH release in OEP rats, suggesting possible excitatory roles for both amines in the regulation of LH secretion. These data also indicate the critical importance of ovarian steroids in determining not only if a neurotransmitter (EPIN) can increase LH release, but also the direction in the LH response to a given neurotransmitter (NE). Finally, in addition to confirming the concept of an excitatory noradrenergic link in the regulation of LH release, studies in nonsteroid primed rats suggest the possible existence of noradrenergic receptors whose activation is subsequently inhibitory to episodic LH secretion.

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. The study was performed in ovariectomized (OVX), ovariectomized estradiol-implanted (OVX + E2) or OVX + E2 progesterone one-treated rats (OVX + E2 + P). The animals were killed before or during the circadian luteinizing hormone (LH) surge. The amount of LHRH-like immunoreactivity recovered from the synaptosomal fraction was slightly increased in OVX + E2-implanted animals but very markedly augmented in OVX + E2 + P-treated rats. In contrast, the higher molecular form recovered from a high-speed supernatant was markedly decreased in OVX + E2 + P-treated rats when compared to the other groups. At the time of maximal LH release induced by E2 + P administration, hypothalamic LHRH was markedly depleted, whereas the larger molecular form was notably augmented. The data suggest that ovarian steroids not only influence release of hypothalamic LHRH but also the processing of LHRH precursor forms.

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. Methylated phospholipids were separated by TLC. Formation of phosphatidyl-mono- and dimethylethanolamine and that of phosphatidylcholine increased significantly in the morning, reaching maximal values on the afternoon of proestrus; they decreased thereafter during estrus, metestrus, and diestrus. Plasma estradiol concentrations increased in late diestrus and then varied similarly with the fluctuations of phospholipid methyltransferase activity throughout the cycle. In parallel, plasma levels of LH and PRL were significantly elevated during the afternoon of proestrus, but remained low throughout the rest of the cycle. Under the same experimental conditions, phospholipid methylation in membranes prepared from mediobasal-hypothalamic structures was not affected. These data demonstrate that under physiological conditions the increased pituitary methyltransferase activity is associated with the progressive increment of plasma estradiol levels occurring shortly before proestrus and precedes the release of LH and PRL. Ovariectomy significantly decreased methyltransferase activity; however, 17 beta-estradiol treatment of ovariectomized rats for 5 days restored the enzyme activity, which was further augmented after progesterone administration. Attempting to investigate variations of pituitary methyltransferase activity in male rats, we demonstrated that the intact males showed weaker activity than that of females; orchidectomy diminished the phospholipid methylation, but adrenalectomy had no effect.

Several neuropeptides have recently been identified within the central nervous system (see review in Snyder, 1980; Guillemin, 1978). Most of them are highly concentrated in hypothalamic areas important for neuroendocrine control (Vale et al., 1980; Elde and Hokfelt, 1978). Overlapping anatomical distributions (Hokfelt et al., 1980; Elde and Hokfelt, 1978; Fuxe, 1965) of several neuropeptides and neurotransmitters within these hypothalamic areas made it tempting to investigate neurotransmitter-neuropeptide interactions and their possible involvement in the adenohypophy-seal regulation (Kordon et al., 1980; Weiner and Ganong, 1978; Schally, 1978).

Publisher Summary This chapter discusses the new designs in neuroendocrine systems. The groups of neurohormones co-localize in neurosecretory neurons, and, in some cases, are co-released into the portal system, so that pituitary cells only see them together. In addition, many receptors are present on different pituitary cell types and trigger more than one second messenger. Most signals that reach the pituitary are mediated by receptors coupled with adenylate cyclase or phospholipases, a category of enzymes that processes biologically important membrane phospholipid derivatives. Presynaptic inhibition by opiates accounts at least in part for these effects. The release of all hypothalamic neuropeptides and that of dopamine from the tuberoinfundibular system is blocked by those peptides. The cell specificity of interactions among coupling chains can account for the target selectivity of combined signals. Within such combinations, some signals carry the biological message itself while others are involved in message addressing, that is, in subselecting targets sharing a common expression of receptors for the same elementary signals.

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. Thus DA inhibits, whereas VIP stimulates adenylate cyclase activity of the same cell type, which, according to the effect of these neurohormones on prolactin secretion, appear to be lactotrophs. On the other hand, SRIF inhibits, whereas GRF stimulates the adenylate cyclase activity of another cell type, namely somatotrophs, whereas CRF appears to act on a third cell type, corticotrophs. Peripheral hormones have been shown to modulate the sensitivity of anterior pituitary cells to these neurohormones. Estradiol long-term treatment has an anti-dopaminergic effect on prolactin secretion. The steroid also suppresses the dopamine inhibition of adenylate cyclase. This effect appears selective to the DA inhibition, since AII inhibition of the enzyme is only partially reduced, whereas the somatostatin inhibition is markedly increased. Peripheral hormones seem to affect the sensitivity of adenohypophyseal cells not only by modulating the number of receptors for a given neurohormone but also by interfering with the coupling mechanisms of these receptors. AII and DA inhibit the adenylate cyclase activity of lactotroph cells. The prolactin stimulation induced by angiotensin is not consistent with the effect of the peptide on adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Somatostatin, in concentrations ranging from 10−10 M to 10−7 M, induces a dose-dependent inhibition of LHRH release from mediobasal hypothalamic slices incubated in vitro. In contrast, VIP, secretin, glucagon, substance P, neurotensin and arginine-vasotocin do not affect spontaneous release of LHRH.

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. Direct addition to the superfusion medium of 17 beta-estradiol (10(-10) to 10(-7) M) or stilbestrol (10(-8) M) potentiated the K+-induced LH-RH release from slices of OVX animals. The K+-induced LH-RH release observed after in vivo E2 implantation was not further amplified by in vitro addition of the hormone. Tamoxifen and hydroxytamoxifen, estrogen antagonists, were ineffective by themselves, but reversed the E2 facilitation of K+-evoked LH-RH release. In contrast, 17 alpha-estradiol, progesterone, or cholesterol (10(-8) or 10(-9) M) hat no effect on either basal or stimulated release of the neurohormone. Somatostatin release measured under identical conditions was not affected by castration or by in vitro addition of the steroid.(1) estradiol appears selectively and specifically involved in the process coupling, nerve endings depolarization, and LH-RH release, and (2) the effect is receptor-mediated and does not appear to require nuclear translocation of the steroid or transcription processes, since it can be readily elicited upon addition of the hormone to nerve endings disconnected from their cell bodies.

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. All these effects of the steroid were dose dependent and specific, as neither 17 alpha-estradiol, dexamethasone, nor progesterone used at the same concentration (10(-9) M) was effective. Furthermore, the modulation of DA-sensitive adenylate cyclases by the steroid required long-term exposure of living cells to 17 beta-estradiol since neither 17 beta-estradiol pretreatment for 4 h nor its addition to broken cells directly into the adenylate cyclase assay induced any alteration in the DA-sensitive adenylate cyclase activity. These results are in agreement with a genomic effect of the steroid. Using both anterior pituitary cells and striatal neurons in culture, 17 beta-estradiol affected neither the total number of DA (D1 and D2) receptors nor the estimated number of adenylate cyclase catalytic units. Therefore, it is suggested that the steroid modifies the coupling process by a mechanism that still has to be elucidated. These results demonstrate an effect of 17 beta-estradiol on DA target cells in both systems.(ABSTRACT TRUNCATED AT 250 WORDS)

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. This interpretation is confirmed by the presence of the same proteins in cells of the corticotroph-like AtT-20/D16 cell line and the regulation of their phosphorylation by CRF. The same phosphoproteins were also detected in non dissociated anterior pituitary tissue from ovariectomized rats. Their phosphorylation was regulated by various hormones and other extracellular agents in a way similar to their regulation in anterior pituitary cells in culture. A 5-day estradiol implant pretreatment of the ovariectomized rats, which stimulates the anterior pituitary gland both directly and indirectly, resulted in a very high level of basal phosphorylation of proteins 1-15. Only very little further stimulation was achieved by the addition of exogenous hypothalamic hormones, indicating that the actual physiological regulatory pathways are the same as those unraveled in the various cell culture model systems. In conclusion, phosphorylation of proteins 1-15 1) can be related to the multihormonal regulation of the various anterior pituitary cell types in culture and 2) corresponds to intracellular molecular mechanisms actually involved in the physiological regulations of pituitary functions in the intact anterior pituitary gland.

In order to investigate the effect of opiates on luteinizing hormone-releasing hormone (LHRH) and somatostatin (SRIF) release, mediobasal hypothalamic (MBH) slices of male adult rats were superfused a

THE morphinomimetic pentapeptides enkephalins1 have been identified in the central nervous system (CNS)2–5. In the rat, Met-enkephalin seems to be the predominant opiate-like pentapeptide6; it has been found in different structures of the brain, including the anterior and the mediobasal hypothalamus (MBH)2–4. Enkephalins, like morphine, have been shown to enhance the secretion of prolactin7–9 and growth hormone9,10, and to inhibit the release of thyroid stimulating hormone11, and luteinising hormone (LH) and ovulation12,13. These effects can be inhibited by naloxone, an opiate antagonist; most authors have postulated that they involve a direct action at the hypothalamic level8–14. In this report, we rule out a direct action of Met-enkephalin on LH releasing hormone (LHRH) release and postulate instead that Met-enkephalin modulates a dopamine-stimulated secretion.