Further evidence for inhibition of episodic luteinizing hormone release in ovariectomized rats by stimulation of dopamine receptors.

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.

Catecholamine involvement in episodic luteinizing hormone release in adult ovariectomized rats.

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.

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. 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.

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. 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.

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. Their effects were inhibited either in PKC-depleted alphaT3-1 cells, or by the PKC inhibitor staurosporine. In the context of the known implication of PKC in LHRH-induced signal transduction, our results show that stathmin phosphorylation is involved in LHRH transduction, either as a result of direct activation of specific PKC isoforms or through a pathway involving kinases downstream to PKC activation.

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. 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.

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. 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.

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. 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.

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. 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.

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. immunoreactivity was progressively increased in cells and media during cell culture. [Hyp9]LHRH and its two smallest precursor forms ([Hyp9]LHRH-(Gly11) 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. [Hyp9]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 Gln1 in pyroGlu1 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 [Hyp9]LHRH shared the same receptor for their biological activity, as assessed by measuring luteinizing hormone release and activation of phospholipase C and A2. [Hyp9]LHRH was, however, less potent than LHRH.

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. 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. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). 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 alphaT3-1 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.

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. 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.

alpha 1-adrenergic receptor involvement in the LH surge in ovariectomized estrogen-primed rats.

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.

Further evidence for the existence of opiate binding sites on neurosecretory LHRH mediobasal hypothalamic terminals.

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-decarboxylase activity remained unchanged. These data indicate that presynaptic opiate binding sites are present on LHRH and SRIF mediobasal hypothalamic nerve endings.

[In vitro study of the interaction between neuromediators and neuropeptides involved in hypothalamic neurosecretion control (author's transl)]. / Interactions entre neuromédiateurs et neuropeptides impliqués dans le contrôle de la neurosécrétion hypothalamique in vitro.

Luteinizing hormone-releasing hormone (LHRH) and somatostatin (SRIF) release was assessed in superfused slices of mediobasal hypothalamus. Release of both neurohormones by depolarizing agents (K+, 56mM ; veratridine, 50 muM) was shown to be Ca2+-dependent, according with the stimulus-secretion coupling hypothesis. Opiates (beta endorphin, 10(-7)M and D-ALA2-Met-enkephalinamide 10(-7)M) did not alter the spontaneous release of LHRH and SRIF, but inhibited significantly the K+-induced neuropeptide release. The effect was reversed by the opiate antagonist naloxone (10(-7)M), while naloxone was ineffective by itself. Vasoactive intestinal peptide (VIP 10(-9)M) significantly inhibited K+ evoked release of SRIF ; LHRH release was unaffected. The effect of VIP on SRIF release was dose-dependent ; secretin, a partial VIP agonist, was also active at higher doses. The data suggest that : 1) opiates, acting through specific opiate receptors located on LHRH and SRIF neurons, modulate the release of the neurohormones ; 2) the inhibitory effect of opiates could be due to an inhibition of calcium influx through voltage-dependent calcium channels ; 3) this interaction may account for the stimulation of growth hormone and the inhibition of luteinizing hormone observed after systemic administration of opiates ; 4) VIP inhibits SRIF release, by acting on VIP receptors present on MBH SRIF terminals ; the effect is consistent with the stimulation of GH reported after in vivo administration of the peptide.

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. These interactions involve discrete actions on second messenger systems; similar processes also account for brain and endocrine effects on immune cells.

Effect of intraventricular infusion of catecholamines on luteinizing hormone release in ovariectomized and ovariectomized, steroid-primed rats.

This study examined alterations in episodic LH release in response to prolonged, slow infusions of dopamine (DA), norepinephrine (NE), or epinephrine (EPIN) into the thire 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--1 1/2 h prior to infusion, 1--1 1/2 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 microliter/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 micrograms/h) or NE (0.3--0.6 micrograms/h), had no effect on episodic LH release or mean blood LH levels. However, administration of 8.5 and 17 micrograms DA/h, and 5.5 and 11 micrograms 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 micrograms EPIN/h had no effect on blood LH levels in uprimed rats. In OVX rats primed 3 days prior to infusion with 50 micrograms 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 micrograms NE/h, with increases in LH relase occurring during infusion. EPIN, in doses ineffective in unprimed rats (5.7 and 11.5 micrograms/h), also caused elevations in blood LH levels in EOP rats. Finally, in rats primed with 5 micrograms 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.

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. Omission of Ca++ in the medium, or addition of a Ca++ channel blocker (D-600, 10(-4) M), of a calmodulin inhibitor (trifluoperazine, 30 microM) or of a calmodulin-dependent tubulin kinase inhibitor (phenytoin, 50 microM), antagonized the stimulatory effect of progesterone. When sodium channels were blocked by tetrodotoxin (5 X 10(-7) M), the stimulatory effect of the steroid was completely abolished. The amplitude of the K+-induced LHRH release was slightly increased in the presence of progesterone (10(-7) M) but only from MBH slices of OVX-E2-treated rats. These results indicate that the secretory response of LHRH to progesterone requires priming with estradiol, is Ca++-dependent and involves mediation of calmodulin and a calmodulin-dependent kinase system.