E-box regulation of gonadotropin-releasing hormone (GnRH) receptor expression in immortalized gonadotrope cells.

The pituitary gland's ability to respond to the hypothalamic hormone GnRH (gonadotropin-releasing hormone) depends directly on the gonadotrope-specific expression of the GnRH receptor (GnRHR), a G-protein coupled transmembrane protein coded by the GnRHR gene. In the present study, we have investigated the potential regulatory role of seven noncanonical E-box enhancer sequences within the 856bp proximal 5'-flanking region of the mGnRHR gene in regulating transcription. These sequences are known to mediate the action of clock gene proteins on the expression of a diverse array of genes both central and peripheral. In the present studies the expression of all of the cognate clock genes was identified in the alphaT3-1 gonadotrope cell line. Additionally, luteinizing hormone-immunoreactive cells in the adult rodent pituitary gland were also shown to co-express the PERIOD-1 protein. By means of chromatin immunoprecipitation of alphaT3-1 nuclear extracts we were able to capture promoter fragments of the GnRHR and Period-1 genes, indicating that E-boxes in these promoters bind the CLOCK protein. RNA interference experiments with alphaT3-1 cells in which Bmal1 expression was attenuated also confirmed the involvement of E-boxes in transcriptional regulation of the mGnRHR gene. Subsequent luciferase reporter assay experiments with GnRHR constructs possessing intact or mutated E-boxes confirmed the use of these sequences for the regulation of mGnRH-R/luc expression. Transient overexpression of the dominant negative E-box-binding factor CLOCK-Delta19, or the inhibitory clock protein mPER1, markedly reduced CLOCK/BMAL1-driven mGnRH-R/luc expression in a dose-dependent fashion. Our data implicate the clock genes as important factors controlling GnRHR expression in murine gonadotrope cells.

Exploratory investigation of the effect of melatonin and caloric restriction on the temporal expression of murine hypothalamic transcripts.

Circadian rhythms of behaviour and gene expression are coupled to endogenous neuronal oscillators located in the hypothalamic suprachiasmatic nuclei (SCN), which are synchronised by the environmental light cycle. Besides light, other factors such as the pineal hormone melatonin, temperature and feeding have entraining properties. During senescence, the circadian system becomes weaker and susceptible to desynchronisation. It is unknown to what extent age-related changes are the result of the deterioration of the hypothalamic master clock. Supplementing ageing mice with melatonin as well as maintaining them on a hypocaloric diet extends the life span and delays age-related diseases. By means of DNA microarrays and the quantitative polymerase chain reaction, we have conducted an exploratory study to compare the effect of long-term melatonin substitution (MEL) and caloric restriction (CR) on circadian gene expression in hypothalamic samples, which contained the SCN as well as other important nuclei involved in nutrient balance, reproduction, and so on. Over 4% of the hypothalamic transcripts showed an overt circadian rhythm in expression, and many of these contain E boxes in their promoter regions, suggesting a direct regulation by circadian clock genes. MEL and CR significantly influenced some of these rhythmically expressed transcripts, but often in opposite ways. Importantly, our studies emphasise that the apparent direction of treatment effects (i.e. up-regulation versus down-regulation) depends on the time of day at which the samples are compared.

Prophylactic melatonin significantly reduces Alzheimer's neuropathology and associated cognitive deficits independent of antioxidant pathways in AßPP(swe)/PS1 mice.

BACKGROUND: Alzheimer's disease (AD) underlies dementia for millions of people worldwide, and its occurrence is set to double in the next 20 years. Currently, approved drugs for treating AD only marginally ameliorate cognitive deficits, and provide limited symptomatic relief, while newer substances under therapeutic development are potentially years away from benefiting patients. Melatonin (MEL) for insomnia has been proven safe with >15 years of over-the-counter access in the US. MEL exerts multiple complementary mechanisms of action against AD in animal models; thus it may be an excellent disease-modifying therapeutic. While presumed to provide neuroprotection via activation of known G-protein-coupled melatonin receptors (MTNRs), some data indicate MEL acts intracellularly to protect mitochondria and neurons by scavenging reactive oxygen species and reducing free radical formation. We examined whether genetic deletion of MTNRs abolishes MEL's neuroprotective actions in the AßPP(swe)/PSEN1dE9 mouse model of AD (2xAD). Beginning at 4 months of age, both AD and control mice either with or without both MTNRs were administered either MEL or vehicle in drinking water for 12 months. RESULTS: Behavioral and cognitive assessments of 15-month-old AD mice revealed receptor-dependent effects of MEL on spatial learning and memory (Barnes maze, Morris Water Maze), but receptor-independent neuroprotective actions of MEL on non-spatial cognitive performance (Novel Object Recognition Test). Similarly, amyloid plaque loads in hippocampus and frontal cortex, as well as plasma Aß1-42 levels, were significantly reduced by MEL in a receptor-independent manner, in contrast to MEL's efficacy in reducing cortical antioxidant gene expression (Catalase, SOD1, Glutathione Peroxidase-1, Nrf2) only when receptors were present. Increased cytochrome c oxidase activity was seen in 16 mo AD mice as compared to non-AD control mice. This increase was completely prevented by MEL treatment of 2xAD/MTNR+ mice, but only partially prevented in 2xAD/MTNR- mice, consistent with mixed receptor-dependent and independent effects of MEL on this measure of mitochondrial function. CONCLUSIONS: These findings demonstrate that prophylactic MEL significantly reduces AD neuropathology and associated cognitive deficits in a manner that is independent of antioxidant pathways. Future identification of direct molecular targets for MEL action in the brain should open new vistas for development of better AD therapeutics.

Bovine pinealocytes in monolayer culture: studies on the adrenergic regulation of melatonin secretion.

Most of what is known regarding adrenergic control of mammalian pineal functions has been derived from investigations with rodents. In an effort to provide a new animal model for pineal research, the bovine pineal gland was selected for practical and theoretical reasons. Bovine pinealocytes in monolayer culture were first characterized in terms of optimal culture conditions, i.e. medium requirements, cell density, preincubation duration and stimulation times. In Dulbecco's minimum essential medium/Ham's F12 medium, 2 x 10(5) cells per well preincubated for 6 days and stimulated for 8 h resulted in large increases in melatonin (mel) release (15- to 17-fold) after adrenergic stimulation. Likewise, cAMP accumulation within 10 min of stimulation increased 4- to 8-fold. (Bu)2cAMP (10(-5)-10(-3) M) produced a significant elevation in MEL secretion. Neither adrenergic dose-response studies, adrenergic antagonist studies nor experiments in which alpha- and beta-agonists were added together provided any evidence for a positive alpha/beta synergism on either MEL release or cAMP accumulation by bovine pinealocytes. This culture system should prove useful for identifying species differences in the signal transduction mechanisms underlying the activation of MEL secretion in the mammalian pineal.

C-type natriuretic peptide (CNP) in the pituitary: is CNP an autocrine regulator of gonadotropes?

Natriuretic peptides act via receptors with intrinsic guanylate cyclase activity to stimulate cGMP production and are thought to be important regulators of neuroendocrine systems. C-Type natriuretic peptide (CNP) is of particular interest in this regard because the highest tissue concentrations of CNP occur in the anterior pituitary, where it is a highly potent stimulator of cGMP production. Here we show that pituitaries of rats and mice contain abundant CNP prohormone messenger RNA (mRNA), but no atrial natriuretic peptide or B-type natriuretic peptide prohormone mRNAs. Using reverse transcriptase-polymerase chain reaction, both A- and B-type natriuretic peptide receptor (GC-A and GC-B, respectively) transcripts were detected in rat and mouse pituitaries, although only the GC-B mRNA was measurable by Northern blotting. Immunohistochemistry revealed CNP-positive cells in the anterior, but not posterior, pituitaries of rats, and the vast majority of these cells were identified as gonadotropes by colocalization of CNP and LH immunoreactivities. Targeted toxicity using GnRH conjugated to the ricin-A chain was used to test whether gonadotropes are also direct targets for GnRH action. The conjugate dose dependently inhibited the proliferation of alpha T3-1 cells (gonadotrope-derived cells with GnRH receptors), but had no such effect on GH3 cells (which do not have GnRH receptors). Culture of rat pituitary cells with the conjugate caused comparable reductions in CNP-stimulated cGMP production, GnRH-stimulated LH release, and CA2+ ionophore (A23187)-stimulated LH release, but did not measurably alter cAMP production in response to pituitary adenylate cyclase-activating polypeptide. We conclude that CNP is synthesized in the pituitary, where it is located predominantly in gonadotropes, and GC-B receptors expressed in the pituitary mediate the direct effects of CNP in gonadotropes. Together with the recent demonstration of CNP synthesis and action in alpha T3-1 cells, the data suggest CNP to be a novel autocrine regulator of gonadotropes.

The human myometrium as a target for melatonin.

The circadian timing of spontaneous human deliveries results in births occurring statistically more often during the nocturnal phase of the 24-h cycle. The neuroendocrine mechanisms underlying this physiological phenomenon are not understood. In an effort to test the hypothesis that melatonin may serve as an endocrine signal for coordinating myometrial events in the human, we determined the mRNA expression of both MT1 and MT2 melatonin receptor isoforms in pregnant as well as nonpregnant myometrial biopsies by means of RT-PCR and in situ hybridization histochemistry. Additionally, we could demonstrate specific, high affinity iodomelatonin binding to myometrial tissues of both pregnant and nonpregnant women. Primary cultures of myocytes responded differentially from melatonin in terms of cAMP signaling depending on the reproductive state. These results imply that melatonin may have the potential to modulate myometrial function in the human, a finding that could open up new possibilities for the development of novel therapeutic agents.

Effects of acute tryptophan depletion on nocturnal melatonin secretion in humans.

Measurement of melatonin secretion throughout the night provides an index of net noradrenergic activity mediated by postsynaptic beta-adrenergic receptors in the pineal gland. Reduced melatonin secretion in some patients with depression might be related to reduced net noradrenergic function. However, a dysregulation in serotonin function has also been implicated in the pathophysiology of depression. The essential amino acid tryptophan is the precursor for both serotonin and melatonin production. To determine the effects of serotonin function on nocturnal melatonin secretion, eight healthy volunteers underwent active and sham tryptophan depletion in a randomized, double-blind fashion. Blood samples for melatonin and free and total tryptophan were obtained before and after the depletion. Acute tryptophan depletion decreased free and total plasma tryptophan levels to less than 20% of baseline levels. Melatonin secretion, expressed as area under the curve, was decreased in all eight subjects after tryptophan depletion when compared to sham depletion. These results suggest that reduced plasma tryptophan levels, and presumably brain serotonin concentrations, decrease nocturnal melatonin secretion in humans. Additional studies investigating the relationship between serotonin metabolism and pineal function in humans appear warranted.

A semiquantitative image-analytical method for the recording of dose-response curves in immunocytochemical preparations.

Knowledge about intracellular signal transduction cascades is largely based on investigations of cultured cells whose responses to different stimuli are typically quantified via RIA, ELISA, or immunoblots. These techniques, which require relatively large amounts of biological material, are performed with homogenized cells and therefore do not allow localization of the molecules under investigation. We describe a protocol for recording dose-response curves directly from immunocytochemical preparations using rat pinealocytes as a model system. The cells were exposed to beta-adrenergic stimuli inducing the phosphorylation of the transcription factor CREB (mediated by PKA), an increase in ICER protein levels, and synthesis and release of melatonin. Melatonin concentrations were determined by ELISA. cPKA, phosphorylated CREB, and ICER were demonstrated by immunocytochemistry and immunoblots. Dose-response curves were recorded by measuring the integrated density of the immunoreactive sites with an image analysis program. Dose-response curves from immunoblots and immunocytochemical preparations showed almost identical dynamics, validating the immunocytochemical approach, which minimizes the amount of biological material needed for such studies, allows combined quantification and localization of biomolecules, and may even be more sensitive than immunoblotting.

Natriuretic peptides elevate cyclic 3',5'-guanosine monophosphate levels in cultured rat pinealocytes: evidence for guanylate cyclase-linked membrane receptors.

Cyclic GMP formation in the rat pinealocyte has generally been thought to involve guanylate cyclases (GC) which are activated via GTP-regulatory proteins following beta 1-adrenergic receptor stimulation. Recent studies have also pointed to a cytosolic GC in these cells whose activity can be elevated by nitric oxide donors. Little attention has been paid to the possibility that pinealocytes might express membrane-bound GC in the form of natriuretic peptide receptors. The present report demonstrates functional membrane GC in rat pinealocytes by (1) cross-linking analyses with radiolabelled atrial natriuretic peptide (ANP); (2) reverse transcriptase polymerase chain reaction (RT-PCR) and DNA blot hybridization with probes for both the GC-A and GC-B forms of the natriuretic receptor; and (3) monolayer cell cultures of pinealocytes, which accumulate cGMP in response to ANP and its related peptides. As the role for cGMP in the rat pineal gland does not appear to be directly coupled to the synthesis of melatonin, the natriuretic peptides may have other regulatory functions in this neuroendocrine tissue.

A fusca gene homologue in mammalian tissues: Developmental regulation in the rat testes.

By differential screening of a rat pineal cDNA library we identified earlier a novel transcript having a 57% nucleotide homology and a 45% amino acid identity with a plant fusca-gene (fus6) to which a corresponding human sequence (gps1) has recently been reported. Expression of this mammalian fusca homologue (mfh) was seen in a variety of mammalian tissues, including kidney, pineal and retina, but it was particularly strong in the testes. Northern blot analysis demonstrated that the rat testicular mfh message increases markedly from day 28 onwards. Additionally, by in situ hybridization, mfh was localized primarily to the seminiferous tubules with a stage-dependent distribution pattern, a result which was confirmed by immunohistochemistry with antibodies raised against a synthetic MFH oligopeptide. Western blotting also revealed strong signals of the expected molecular weight in testicular extracts from several species. In view of its homology to fus6, a plant gene known to be involved in repressing photomorphogenesis in darkness, the conservation of mfh in mammals suggests a potential function for MFH in signaling pathways involved in the regulation of mammalian differentiation and development.

Urinary 6-hydroxymelatonin sulfate as a measure of melatonin secretion during acute tryptophan depletion.

The essential amino acid tryptophan (TRP) is the precursor for serotonin (5-HT), which is in turn an intermediary product in the synthesis of melatonin. Acute TRP depletion has recently been shown to decrease nocturnal plasma levels of melatonin in humans. Melatonin is metabolized to 6-hydroxymelatonin sulfate (6-SM), a highly stable end-product which is excreted into urine. To determine the effects of TRP bioavailability on 6-SM, 11 healthy volunteers underwent active and sham TRP depletion in a randomized, double-blind fashion. Samples of plasma free and total TRP, plasma melatonin, and urinary 6-SM were obtained before and after the depletion. Acute TRP depletion decreased free and total plasma tryptophan levels by more than 80% from baseline levels. Nocturnal 6-SM excretion was significantly decreased and highly correlated with decreases in plasma melatonin. These results suggest that nocturnal urinary 6-SM excretion is a valid measure of melatonin secretion under conditions of decreased 5-HT function. Collection of urine for 6-SM is considerably easier than nocturnal plasma sampling for melatonin. Further studies are needed to clarify the relationship between 6-SM excretion and other measures of 5-HT function in neuropsychiatric disorders.

Guanylyl cyclase-B represents the predominant natriuretic peptide receptor expressed at exceptionally high levels in the pineal gland.

The generation and function(s) of the signalling molecule cyclic GMP (cGMP) in brain are still poorly understood. One mechanism to raise intracellular cGMP levels is binding of C-type natriuretic peptide (CNP) to a membrane guanylyl cyclase (GC), termed GC-B. Here, we demonstrate an exceptionally strong expression of GC-B in the pineal gland. Crosslinking experiments performed with 125I-Tyr(0)-CNP and membranes from various rat tissues identified the receptor as a 130-kDa protein, expressed at highest levels in pineal membranes. Receptor autoradiography on brain sections confirmed a striking density of CNP binding sites in pineal tissue, whereas binding sites for the related atrial natriuretic peptide (ANP) predominate in other regions of the brain. Incubations of freshly dissected whole pineal glands in either the absence or presence of natriuretic peptides followed by immunohistochemical analyses of cGMP revealed strong accumulations of cGMP in response to CNP but not to ANP in the majority of pinealocytes. Stimulation of soluble GC (sGC) activity by use of sodium nitroprusside (SNP) resulted in a very similar pattern of cGMP immunostaining, indicating a co-expression at high levels of particulate and soluble forms of GC. These findings point to a major role of cGMP signalling in pinealocytes and suggest an important regulatory function for CNP.

The effect of NO-donors in bovine and rat pineal cells: stimulation of cGMP and cGMP-independent inhibition of melatonin synthesis.

The presence of soluble guanylate cyclase in the pineal and its regulation by adrenergic pathways has been well documented. Recent evidence points to adrenergically stimulated nitric oxide generation as a mechanism for coupling this pathway. To what extent nitric oxide (NO) signalling can influence adrenergically stimulated melatonin synthesis has not been investigated. Cyclic guanosine 3',5'-monophospate (cGMP) signal transduction in the bovine pineal has also received little attention. We describe in the present report: 1) a dose-dependent elevation of cGMP in response to the nitrovasodilators, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1), 2) a dose-dependent inhibition of melatonin synthesis by SNP and SIN-1, but not by 8-Br-cGMP in both bovine and rat pineal cell cultures, which is not due to cytotoxicity as judged by two different approaches, and 3) immunohistochemical evidence for the presence of nitric oxide synthase (NOS) (EC 1.14.23.-) in the intact bovine pineal gland and in cultured bovine pinealocytes. These data support the view that NOS is a component of the cGMP-generating system in mammalian pinealocytes. Although NO-donor molecules are also potent activators of cGMP accumulation, they may have other important actions in the pineal, namely the inhibition of adrenergic-stimulated melatonin synthesis. As SNP and SIN-1 exerted this inhibitory effect on cells regardless of whether they were stimulated by isoproterenol, forskolin or 8-Br-cAMP it would appear that NO-donors can act 'downstream' from the receptor/adenylate cyclase level.

Nocturnal accumulation of cyclic 3',5'-guanosine monophosphate (cGMP) in the chick pineal organ is dependent on activation of guanylyl cyclase-B.

The role of cGMP in the avian pineal is not well understood. Although the light-sensitive secretion of melatonin is a well-known output of the circadian oscillator, pharmacologically elevated cGMP levels do not result in altered melatonin secretory amplitude or phase. This suggests that pineal cGMP signalling does not couple the endogenous circadian oscillator to the expression of melatonin rhythms. Nonetheless, the free-running rhythm of cGMP signalling implies a link to the circadian oscillator in chick pinealocytes. As the circadian rhythm of cGMP levels in vitro is not altered by pharmacological inhibition of phosphodiesterase activity, we infer that the synthesis, rather than the degradation of cGMP, is under circadian control. In vitro experiments with the nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine as well as with an inhibitor of the NO-sensitive soluble guanylyl cyclase (sGC), showed that the NOS-sGC pathway does not play a major role in the circadian control of cGMP generation. In organ culture experiments, we demonstrated that C-type natriuretic peptide (CNP), but not atrial natriuretic peptide (ANP), elevated daytime levels of cGMP. As CNP acts on the membrane guanylyl cyclase isoform B (GC-B), which is expressed at very high levels in mammalian pineals, we investigated the effect of the membrane GC-specific inhibitor HS-142 on chick pineal cGMP levels. CNP-induced daytime cGMP levels were reduced by HS-142. More importantly, 'spontaneously' high nocturnal levels of cGMP in vitro were reduced to daytime levels by acute addition of HS-142. These data implicate endogenous nocturnal CNP release and subsequent activation of GC-B as the major input driving cGMP synthesis in the chick pineal organ.

Natriuretic peptides stimulate cyclic GMP production in an immortalized LHRH neuronal cell line.

The role of cyclic 3',5'-guanosine monophosphate (cGMP) as a second messenger in LHRH neurons is not well understood. Recent studies involving nitric oxide, a direct activator of soluble guanylate cyclase (GC), have implicated cGMP in the regulation of LHRH secretion both in vivo and in vitro. Evidence for the membrane-bound form of GC in LHRH neurons has thus far not been reported. In polymerase chain reaction screening of various cell lines for the natriuretic peptide receptors--which represent GCs--we identified both GC-A and GC-B cDNAs by southern blot hybridization in reverse transcribed and amplified extracts of the GT1-7 cell line, an immortalized LHRH neuronal cell line. Subsequent experiments demonstrated that all of the natriuretic peptides elevated cGMP production with a rank order of potency: CNP > ANP > BNP. Time course studies revealed a rapid intracellular accumulation of cGMP following exposure to CNP with a peak at 2.5 min. CNP was some 200-fold more potent than the NO donor, sodium nitroprusside, in stimulating cGMP accumulation in these cells. These data show for the first time the presence of functional mGCs on LHRH cells, and suggest that the natriuretic peptides may also participate in the regulation of LHRH activity.

Pituitary adenylate cyclase-activating peptide and vasoactive intestinal peptide receptor expression in immortalized LHRH neurons.

The regulation of LHRH secretion is extraordinarily multifarious. To no small extent, this insight has been gained through studies using the immortalized hypothalamic LHRH neuronal line, GT1-7. In the present study, we examined these cells for potential expression of the receptors for the related peptides PACAP and VIP. By means of reverse transcription-polymerase chain reaction (RT-PCR) with PACAP receptor-specific primers, in combination with restriction enzyme analysis and cDNA sequencing, we were able to identify all PACAP-specific receptor splice variant forms with variable degrees of expression. Of the two nonselective VIP/PACAP receptors (i.e. VIP-R type I and II) only the latter isoform was detected by RT-PCR. In view of these results, we sought to establish whether PACAP and VIP receptors are functional in GT1-7 cells. Cyclic AMP (cAMP) accumulation after addition of PACAP-38 (or PACAP-27) was dose-dependent with maximal 3-fold increases. VIP also elevated cAMP with a similar potency. Phosphatidylinositol (PI) turnover was unaffected by either PACAP or VIP. Acute LHRH secretion was stimulated equally by nanomolar concentrations of both PACAP and VIP. These results point to PACAP and VIP having direct actions via the VIP2R on cAMP signalling and LHRH release, in addition to the known effects of these peptides on pituitary functions.

Synthesis of C-type natriuretic peptide (CNP) by immortalized LHRH cells.

Former studies have indicated an influence of natriuretic peptides on LHRH secretion. In this report we demonstrate local synthesis of CNP in immortalized LHRH neurons (GT1-7 cells). Using reverse transcription-polymerase chain reaction and RNase protection assays a transcript for the CNP precursor was identified in these cells. Immunocytochemical data revealed the presence of the peptide CNP in GT1 cells, using a specific polyclonal antiserum against CNP. Electron microscopic immunohistochemical investigations also showed the strongest CNP-immunoreactivity in some small vesicles, providing initial evidence for the potential secretion of this peptide by immortalized LHRH neurons. Subsequent experiments demonstrated also that CNP elevates LHRH production in static cultures of GT1 cells. These data show for the first time the co-production of the functionally relevant natriuretic peptide, CNP, by immortalized LHRH neurons. Together with the recent demonstration of CNP receptor expression by these cells, we suggest that CNP may represent a novel autocrine regulator of LHRH neuronal activity. It remains to be elucidated, however, to what extent CNP expression in immortalized LHRH neurons reflects a co-localization in situ of CNP and LHRH peptides.

Pituitary adenylate cyclase activating peptide-38 (PACAP-38), PACAP-27, and PACAP related peptide (PRP) in the rat median eminence and pituitary.

Pituitary adenylate cyclase activating peptide (PACAP) is a member of the vasoactive intestinal peptide-like peptide family. It is found in the hypothalamus, where the PACAP precursor is processed to form PACAP-38, the C-terminal truncated PACAP-27 and PACAP related peptide (PRP). Both PACAPs are potent stimulators of anterior pituitary adenylate cyclase activity, but the physiologically relevant anatomical sources of PACAP and possible importance of PRP in this regard are poorly understood. Using immunocytochemistry with epitope-specific antisera, we now show that PACAP38-, PACAP27- and PRP-positive nerve fibres are all present in the rat median eminence. The major immunoreactive species present was PACAP38. Numerous PACAP38-immunoreactive nerve fibres were observed in the internal layer and a few were present in the posterior pituitary lobe. The external layer of the median eminence contained a few PACAP-38-immunoreactive fibres and PACAP-38-positive nerve terminals were rarely seen in the perivascular portal spaces. Surprisingly, delicate PACAP-38-positive nerve fibres were identified in the anterior pituitary lobe intermingled between the pituitary cells although none of the secretory pituitary cells contained immunoreactive PACAP38, PACAP27 or PRP and preproPACAP mRNA was not detected in the gland by Northern blotting or in situ hybridization. PACAP-27- and PRP-immunoreactive nerve fibres and terminals were found in the same locations as PACAP-38 although generally in lower numbers. Specific radioimmunoassays and HPLC revealed that PACAP-38 accounts for the vast majority of the adenohypophyseal PACAP-immunoreactivity, whereas PACAP-27 and PRP were found in low to undetectable concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and regulation of mPer1 in immortalized GnRH neurons.

Hypothalamic GnRH (gonadotropin-releasing hormone) neurons play a critical role in the initiation and maintenance of reproduction competence. Using the mouse GnRH neuronal cell line, GT1-7, we have characterized the expression of the gene mPer1, a recognized key element of the mammalian circadian clockwork. Both mPer1 transcripts and the 136 kDa mPER1 gene product could be detected in these cells. Immunocytochemical analysis also confirmed expression of mPER1 both in vitro and in vivo in GnRH neurons. Activation of cyclic AMP signalling pathways in vitro elevated GnRH secretion as well as mPer1 expression and nuclear mPER1 immunoreactivity. As mPER1 is known to feedback on transcriptional activities in many cell models, the data presented here point to a role for mPER1 in the regulation of gene expression in GnRH neurons, and thus in the control of neuroendocrine activities.

Extracellular serotonin promotes melatonin release from cultured rat pinealocytes: evidence for an S2-type receptor-mediated autocrine feedback.

Recent evidence points to the secretion of serotonin from the rat pineal gland both in vivo and in vitro. In view of the fact that adrenergic stimulation of cultured pinealocytes leads to rapid serotonin release well in advance of melatonin production, the question arises as to the physiological significance of serotonin secretion. The present studies examined the impact of ketanserine, a specific serotonin type 2 receptor antagonist, on the cyclic AMP and melatonin response of cultured rat pinealocytes to adrenergic stimuli. Whereas the beta-adrenergic agonist isoproterenol (1 microM) stimulated cAMP accumulation as well as melatonin release significantly, the presence of ketanserine inhibited these responses in a dose dependent manner. Since the same inhibitory effect of ketanserine was seen in cells stimulated by dibutyryl-cAMP (which acts post-adrenergic receptor to elevate melatonin synthesis), the mechanism for serotonin's feedback actions does not appear to involve changes in adrenergic receptor/cAMP coupling. These results indicate that extracellular serotonin may be important for the full activation of melatonin secretion following adrenergic stimulation.