Hypophysectomy abolishes rhythms in rat thyroid hormones but not in the thyroid clock.

The endocrine body rhythms including the hypothalamic-pituitary-thyroid axis seem to be regulated by the circadian timing system, and daily rhythmicity of circulating thyroid-stimulating hormone (TSH) is well established. The circadian rhythms are generated by endogenous clocks in the central brain oscillator located in the hypothalamic suprachiasmatic nucleus (SCN) as well as multiple peripheral clocks, but information on the existence and function of a thyroid clock is limited. The molecular machinery in all clock cells is composed of a number of clock genes and their gene products are connected by autoregulatory feedback loops. Here, we provide evidence for a thyroid clock in the rat by demonstrating 24-h antiphase oscillations for the mRNA of the canonical clock genes Per1 and Bmal1, which was unaffected by hypophysectomy. By immunostaining, we supported the existence of a core oscillator in the individual thyroid cells by demonstrating a daily cytoplasmatic-nuclear shuttling of PER1 protein. In normal rats, we found a significant daily rhythmicity in the circulating thyroid hormones preceded by a peak in TSH. In hypophysectomised rats, although the thyroid clock was not affected, the oscillations in circulating thyroid hormones were abolished and the levels were markedly lowered. No daily oscillations in the expression of TSH receptor mRNA were observed in neither control rats nor hypophysectomised rats. Our findings indicate that the daily rhythm of thyroid hormone secretion is governed by SCN signalling via the rhythmic TSH secretion rather than by the local thyroid clock, which was still ticking after hypophysectomy.

Pituitary adenylate cyclase-activating polypeptide promotes eccrine gland sweat secretion.

BACKGROUND: Sweat secretion is the major function of eccrine sweat glands; when this process is disturbed (paridrosis), serious skin problems can arise. To elucidate the causes of paridrosis, an improved understanding of the regulation, mechanisms and factors underlying sweat production is required. Pituitary adenylate cyclase-activating polypeptide (PACAP) exhibits pleiotropic functions that are mediated via its receptors [PACAP-specific receptor (PAC1R), vasoactive intestinal peptide (VIP) receptor type 1 (VPAC1R) and VPAC2R]. Although some studies have suggested a role for PACAP in the skin and several exocrine glands, the effects of PACAP on the process of eccrine sweat secretion have not been examined. OBJECTIVES: To investigate the effect of PACAP on eccrine sweat secretion. METHODS: Reverse transcriptase-polymerase chain reaction and immunostaining were used to determine the expression and localization of PACAP and its receptors in mouse and human eccrine sweat glands. We injected PACAP subcutaneously into the footpads of mice and used the starch-iodine test to visualize sweat-secreting glands. RESULTS: Immunostaining showed PACAP and PAC1R expression by secretory cells from mouse and human sweat glands. PACAP immunoreactivity was also localized in nerve fibres around eccrine sweat glands. PACAP significantly promoted sweat secretion at the injection site, and this could be blocked by the PAC1R-antagonist PACAP6-38. VIP, an agonist of VPAC1R and VPAC2R, failed to induce sweat secretion. CONCLUSIONS: This is the first report demonstrating that PACAP may play a crucial role in sweat secretion via its action on PAC1R located in eccrine sweat glands. The mechanisms underlying the role of PACAP in sweat secretion may provide new therapeutic options to combat sweating disorders.

Altered Expression Pattern of Clock Genes in a Rat Model of Depression.

BACKGROUND: Abnormalities in circadian rhythms may be causal factors in development of major depressive disorder. The biology underlying a causal relationship between circadian rhythm disturbances and depression is slowly being unraveled. Although there is no direct evidence of dysregulation of clock gene expression in depressive patients, many studies have reported single-nucleotide polymorphisms in clock genes in these patients. METHODS: In the present study we investigated whether a depression-like state in rats is associated with alternations of the diurnal expression of clock genes. The validated chronic mild stress (CMS) animal model of depression was used to investigate rhythmic expression of three clock genes: period genes 1 and 2 (Per1 and Per2) and Bmal1. Brain and liver tissue was collected from 96 animals after 3.5 weeks of CMS (48 control and 48 depression-like rats) at a 4h sampling interval within 24h. We quantified expression of clock genes on brain sections in the prefrontal cortex, nucleus accumbens, pineal gland, suprachiasmatic nucleus, substantia nigra, amygdala, ventral tegmental area, subfields of the hippocampus, and the lateral habenula using in situ hybridization histochemistry. Expression of clock genes in the liver was monitored by real-time quantitative polymerase chain reaction (PCR). RESULTS: We found that the effect of CMS on clock gene expression was selective and region specific. Per1 exhibits a robust diurnal rhythm in most regions of interest, whereas Bmal1 and in particular Per2 were susceptible to CMS. CONCLUSION: The present results suggest that altered expression of investigated clock genes is likely associated with the induction of a depression-like state in the CMS model.

Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase.

Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngb's function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

Neuroglobin expression in the rat suprachiasmatic nucleus: colocalization, innervation, and response to light.

Neuroglobin (Ngb) is a myoglobin-like (Mb) heme-globin, belonging the globin family located only in neuronal tissue of the central nervous system. Ngb has been shown to be upregulated in and to protect neurons from hypoxic and ischemic injury, but the function of Ngb-in particular how Ngb may protect neurons-remains largely elusive. We have previously described the localization of Ngb in the rat brain and found it to be expressed in areas primarily involved in sleep/wake, circadian, and food regulation. The present study was undertaken, using immunohistochemistry, to characterize the localization, colocalization, innervation, and response to light of Ngb-immunoreactive (IR) cells in the rat suprachiasmatic nucleus (SCN). Our results demonstrate that the majority of Ngb-expressing neurons in the SCN belong to a cell group not previously characterized by neurotransmitter content; only a small portion was found to co-store GRP in the ventral SCN. Furthermore, some Ngb-containing neurons were responsive to light stimulation at late night evaluated by the induction of cFOS and only a few cells were found to express the core clock gene PER1 during the 24-hour light/dark cycle. The Ngb-containing cells received input from neuropeptide Y (NPY)-containing nerve fibers of the geniticulo-hypothalamic tract (GHT), whereas no direct input from the eye or the midbrain raphe system was demonstrated. The results indicate that the Ngb could be involved in both photic and nonphotic entrainment via input from the GHT.

Vasoactive intestinal peptide causes marked cephalic vasodilation, but does not induce migraine.

We hypothesized that intravenous infusion of the parasympathetic transmitter, vasoactive intestinal peptide (VIP), might induce migraine attacks in migraineurs. Twelve patients with migraine without aura were allocated to receive 8 pmol kg(-1) min(-1) VIP or placebo in a randomized, double-blind crossover study. Headache was scored on a verbal rating scale (VRS), mean blood flow velocity in the middle cerebral artery (V(mean MCA)) was measured by transcranial Doppler ultrasonography, and diameter of the superficial temporal artery (STA) by high-frequency ultrasound. None of the subjects reported a migraine attack after VIP infusion. VIP induced a mild immediate headache (maximum 2 on VRS) compared with placebo (P = 0.005). Three patients reported delayed headache (3-11 h after infusion) after VIP and two after placebo (P = 0.89). V(mean MCA) decreased (16.3 +/- 5.9%) and diameter of STA increased significantly after VIP (45.9 +/- 13.9%). VIP mediates a marked dilation of cranial arteries, but does not trigger migraine attacks in migraineurs. These data provide further evidence against a purely vascular origin of migraine.

Diurnal rhythmicity of the canonical clock genes Per1, Per2 and Bmal1 in the rat adrenal gland is unaltered after hypophysectomy.

Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus (SCN), and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. Rhythmic expression of clock genes in the adrenal glands has previously been reported. Since the central clock in the SCN communicates with the adrenal glands via circadian release of adrenocorticotrophic hormone, we quantified the mRNAs for the canonical clock genes, Per1, Per2 and Bmal1 in the adrenal glands by real-time reverse transcription-polymerase chain reaction during a 24-h-cycle in normal and hypophysectomised rats. The mRNAs for all the three clock genes disclosed rhythmic oscillations with a period of 24 h and the phase did not differ between the hypophysectomised and intact rats. The expression pattern of Per1 and Bmal1 was in antiphase in both groups of animals. In situ hybridisation histochemistry using antisense RNA probes demonstrated that, at times of peak expression, mRNAs for all the three clock genes were expressed in the adrenal cortex with a particularly strong labelling in the zona reticularis. In accordance with the mRNA localisation, immunostaining for PER1 protein was visualised in cells of the adrenal cortex, being most intense in the inner zone. The immunostaining also demonstrated a translocation of PER1 protein from the cytoplasm to the nucleus during the daily cycle, supporting the existence of a core oscillator in the individual adrenal gland cells. Our findings support the existence of a circadian core oscillator in cells of the rat adrenal cortex and indicate that the activity of the oscillator is independent of SCN signalling via the pituitary gland. The adrenal cortical clock could be involved in rhythmic transcriptional activation of genes associated with hormonal biosynthesis, involved in gating of the response of the adrenal cortex to external cues or involved in apoptosis of adrenal cortical cells.

Vasoactive intestinal polypeptide evokes only a minimal headache in healthy volunteers.

The role of the parasympathetic nervous system in the pathogenesis of migraine is disputed. The headache-eliciting effect of the parasympathetic neurotransmitter, vasoactive intestinal polypeptide (VIP), and its effect on cerebral arteries and brain haemodynamics has not been systematically studied in man. We hypothesized that infusion of VIP might induce headache in healthy subjects and cause changes in cerebral haemodynamics. VIP (8 pmol/kg per min) or placebo (0.9% saline) was infused for 25 min into 12 healthy young volunteers in a crossover, double-blind design. Headache was scored on a verbal rating scale from 0 to 10, regional cerebral blood flow (rCBF) was measured with single-photon emission computed tomography and (133)Xe inhalation and mean flow velocity in the middle cerebral artery (V(meanMCA)) was measured with transcranial Doppler ultrasonography. The headache was very mild with a maximum score of 2 and described as a pressing or throbbing sensation. Five participants developed headache during VIP and one during placebo. During the infusion, a significant drop in V(meanMCA) was seen for VIP compared with placebo (P < 0.001), but the effect quickly waned and no difference was found when comparing the time between 30 and 120 min. In addition, no significant difference in the diameter of the MCA could be found during the infusion. No significant differences in rCBF (P = 0.10) were found between VIP and placebo. A marked dilation of the superficial temporal artery was seen (P = 0.04) after VIP in the first 30 min but no difference was found when comparing the time between 30 and 120 min. We found no difference in mean arterial blood pressure between VIP and placebo days but the heart rate increased significantly on a VIP day compared with a placebo day (AUC(0-30 min), P < 0.001). Plasma VIP was significantly higher on a VIP day compared with placebo (AUC(0-80 min), P < 0.001). These results show that VIP causes a decrease in V(meanMCA) without affecting rCBF. In spite of a marked vasodilator effect in the extracranial vessels and increased plasma VIP, healthy subjects developed only a very mild headache.

Neuronal input pathways to the brain's biological clock and their functional significance.

Circadian rhythms are entrained daily by environmental photic and non-photic cues. The present review describes the anatomy and functional characteristics of the three major input pathways to the circadian clock mediating entrainment: the retino-hypothalamic tract, the geniculo-hypothalamic tract and the midbrain raphe projection.

Expression of pituitary adenylate cyclase activating peptide in the uterine cervix, lumbosacral dorsal root ganglia and spinal cord of rats during pregnancy.

The uterine cervix is highly innervated by the sensory nerves containing neuropeptides which change during pregnancy and are regulated, in part, by estrogen. These neuropeptides act as transmitters both in the spinal cord and cervix. The present study was undertaken to determine the expression pattern of the neuropeptide pituitary adenylate cyclase activating peptide (PACAP) in the cervix and its nerves during pregnancy and the influence of estrogen on this expression using immunohistochemistry, radioimmunoassay and RT-PCR. PACAP immunoreactivity was detected in nerves in the cervix, lumbosacral (L6-S1) dorsal root ganglia (DRG) and spinal cord. PACAP immunoreactivity was highest at day 15 of pregnancy in the cervix and dorsal spinal cord, but then decreased over the last trimester of pregnancy. However, levels of PACAP mRNA increased in the L6-S1 DRG at late pregnancy relative to early pregnancy. DRG of ovariectomized rats treated with estrogen showed increased PACAP mRNA synthesis in a dose-related manner, an effect partially blocked by the estrogen receptor (ER) antagonist ICI 182,780. We postulate that synthesis of PACAP in L6-S1 DRG and utilization in the cervix and spinal cord increase over pregnancy and this synthesis is the under influence of the estrogen-ER system. Since PACAP is expressed by sensory nerves and may have roles in nociception and vascular function, collectively, these data are consistent with the hypothesis that sensory nerve-derived neuronal factors innervate the cervix and play a role in cervical ripening.

Light-induced phase shift in the Syrian hamster (Mesocricetus auratus) is attenuated by the PACAP receptor antagonist PACAP6-38 or PACAP immunoneutralization.

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are daily adjusted (entrained) by light via the retinohypothalamic tract (RHT). The RHT contains two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), which are believed to mediate the phase-shifting effects of light on the clock. In the present study we have elucidated the role of PACAP in light-induced phase shifting at early night in hamsters and shown that (i) light-induced phase delay of running-wheel activity was significantly attenuated by a specific PAC1 receptor antagonist (PACAP6-38) or by immunoblockade with a specific anti-PACAP antibody injected intracerebroventricularly before light stimulation; (ii) PACAP administered close to the SCN was able to phase-delay the circadian rhythm of running-wheel activity in a similar way to light; (iii) PACAP was present in the hamster RHT, colocalized with melanopsin, a recently identified opsin which has been suggested to be a circadian photopigment. The findings indicate that PACAP is a neurotransmitter of the RHT mediating photic information to the clock, possibly via melanopsin located exclusively on the PACAP-expressing cells of the RHT.

Serotonin inhibits glutamate- but not PACAP-induced per gene expression in the rat suprachiasmatic nucleus at night.

Circadian rhythms of physiology and behaviour generated by the brain's biological clock located in the suprachiasmatic nucleus are entrained by light via the retinohypothalamic tract. Two neurotransmitters, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP), found in this monosynaptic pathway mediate the effects of light to the clock. It is well known that not only light entrains the clock. Nonphotic cues mediated by neurotransmitters such as serotonin reaching the suprachiasmatic nucleus from the midbrain raphe nucleus modulate light-induced phase shifts at night. Two clock genes, per1 and per2, have been attributed a role in light-induced phase shift. In the present study, using an in vitro brain slice model and quantitative in situ hybridization for per1 and per2, we have shown that serotonin induces per1 gene expression at late subjective night but not at early night. Furthermore, serotonin application before glutamate or PACAP blocked glutamate-induced per1 expression at early night and per2 gene expression at late night. In contrast, serotonin did not influence PACAP-induced per gene expression at late night. Triple antigen immunohistochemistry and confocal microscopy supported both a pre- and post-synaptic interaction of retinohypothalamic tract (PACAP-immunoreactive) and serotonin projections on vasoactive intestinal peptide- and gastrin-releasing peptide-containing cell bodies in the ventro-lateral suprachiasmatic nucleus. Our findings suggest that the per genes could be the molecular target for the modulatory effects of serotonin on light signalling to the clock.

Gut/brain peptides in the genital tract: VIP and PACAP.

The two structurally related gut/brain peptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are pleiotropic peptides with a wide-spread occurrence. Besides their presence and functions in the gut and the brain VIP and PACAP have distinct physiological roles in the genital tract. VIP seems to be involved in the nervous control of ovum transportation, sexual arousal in women and penile erection in men. Dysfunction of the VIP nerves can lead to erectile failure and VIP in combination with phentolamine can be successfully used as self-injection therapy of impotence. PACAP could be a co-transmitter with VIP in a number of functions involving nervous control of blood flow and motility, but in addition PACAP is a sensory neurotransmitter. The most fascinating role for PACAP is, however, its auto- or paracrine function in the peri- and postovulatory events involving acute progesterone production and subsequent luteinization in periovulatory granulosa/lutein cells.

Light-dependent induction of cFos during subjective day and night in PACAP-containing ganglion cells of the retinohypothalamic tract.

Environmental light stimulation via the retinohypothalamic tract (RHT) is necessary for stable entrainment of circadian rhythms generated in the suprachiasmatic nucleus (SCN). In the current report, the authors characterized the functional activity and phenotype of retinal ganglion cells that give rise to the RHT of the rat. Retinal ganglion cells that give rise to the RHT were identified by transsynaptic passage of an attenuated alpha herpesvirus known to have selective affinity for this pathway. Dual labeling immunocytochemistry demonstrated co-localization of viral antigen and pituitary adenylate cyclase activating polypeptide (PACAP) in retinal ganglion cells. This was confirmed using the anterograde tracer cholera toxin subunit B (ChB). In normal and retinally degenerated monosodium glutamate (MSG)-treated rats, ChB co-localized with PACAP in axons of the retinorecipient zone of the SCN. Light-induced Fos-immunoreactivity (Fos-IR) was apparent in all PACAP-containing retinal ganglion cells and a population of non-PACAP-containing retinal ganglion cells at dawn of normal and MSG-treated animals. Within the next 3 h, Fos disappeared in all non-PACAP-immunoreactive cells but persisted in all PACAP-containing retinal ganglion cells until dusk. When animals were exposed to constant light, Fos-IR was sustained only in the PACAP-immunoreactive (PACAP-IR) retinal ganglion cells. Darkness eliminated Fos-IR in all PACAP-IR retinal ganglion cells, demonstrating that the induction of Fos gene expression was light dependent. When animals were maintained in constant darkness and exposed to light pulses at ZT 14, ZT 19, or ZT 6, Fos-IR was induced in PACAP-IR retinal ganglion cells in a pattern similar to that seen at dawn. Collectively, these data indicate that PACAP is present in ganglion cells that give rise to the RHT and suggest a role for this peptide in the light entrainment of the clock.

Functional roles of conserved transmembrane prolines in the human VPAC(1) receptor.

The importance of three conserved transmembrane prolines of the human vasoactive intestinal polypeptide (VPAC)(1) receptor was examined by single alanine substitution. P266A, P300A and P348A reduced the expression level, but maintained the binding to VIP. P266A showed decreased ability to stimulate cAMP, while P300A and P348A displayed an increased potency in cAMP production combined with a high sensitivity towards GTP compared to the wild type receptor. In addition, substitutions of two conserved leucines located in position -2 and +1 from P348 were investigated. L346A and L349A reduced the receptor expression, influenced the G protein coupling and decreased the receptor activity. These observations, which are the first on conserved transmembrane prolines within this family of receptors, indicate that these residues are important for receptor expression, G protein coupling and receptor activity.

PACAP 1-38 as neurotransmitter in the porcine antrum.

UNLABELLED: The concentration of PACAP 1-38 in porcine antrum amounted to 15.4+/-7.9 and 20.3+/-8 pmol/g tissue in the mucosal and muscular layers. PACAP immunoreactive (IR) fibres innervated the muscular (co-localised with VIP) and submucosal/mucosal layers (some co-storing VIP and CGRP) including myenteric and submucosal plexus and blood vessels. Only myenteric nerve cell bodies contained PACAP-IR (co-storing VIP). In isolated perfused antrum, vagus nerve stimulation (8 Hz) and capsaicin (10(-5) M) increased PACAP 1-38 release. PACAP 1-38 (10(-9) M) increased substance P (SP), gastrin releasing peptide (GRP) and VIP release. PACAP 1-38 (10(-8) M) inhibited gastrin secretion and stimulated somatostatin secretion and motility dose-dependently. PACAP-induced motility was strongly inhibited by the antagonist PACAP 6-38 but also by atropine and substance P-antagonists (CP99994/SR48968) but PACAP 6-38 had no effect on vagus-induced secretion or motility. CONCLUSION: PACAP 1-38 may be involved in antral motility and secretion by interacting with cholinergic, SP-ergic, GRP-ergic and/or VIP-ergic neurones, and may also be involved in afferent reflex pathways.

Dissociation between light-induced phase shift of the circadian rhythm and clock gene expression in mice lacking the pituitary adenylate cyclase activating polypeptide type 1 receptor.

The circadian clock located in the suprachiasmatic nucleus (SCN) organizes autonomic and behavioral rhythms into a near 24 hr time that is adjusted daily to the solar cycle via a direct projection from the retina, the retinohypothalamic tract (RHT). This neuronal pathway costores the neurotransmitters PACAP and glutamate, which seem to be important for light-induced resetting of the clock. At the molecular level the clock genes mPer1 and mPer2 are believed to be target for the light signaling to the clock. In this study, we investigated the possible role of PACAP-type 1 receptor signaling in light-induced resetting of the behavioral rhythm and light-induced clock gene expression in the SCN. Light stimulation at early night resulted in larger phase delays in PACAP-type 1 receptor-deficient mice (PAC1(-)/-) compared with wild-type mice accompanied by a marked reduction in light-induced mPer1, mPer2, and c-fos gene expression. Light stimulation at late night induced mPer1 and c-fos gene expression in the SCN to the same levels in both wild type and PAC1(-)/- mice. However, in contrast to the phase advance seen in wild-type mice, PAC1(-)/- mice responded with phase delays after photic stimulation. These data indicate that PAC1 receptor signaling participates in the gating control of photic sensitivity of the clock and suggest that mPer1, mPer2, and c-fos are of less importance for light-induced phase shifts at night.

Pituitary adenylate cyclase-activating polypeptide induces period1 and period2 gene expression in the rat suprachiasmatic nucleus during late night.

The suprachiasmatic nucleus generates circadian rhythms which are synchronized to the environmental light-dark cycle via the retinohypothalamic tract. Pituitary adenylate cyclase-activating polypeptide and glutamate, two neurotransmitters co-stored in the retinohypothalamic tract of the rat, are able to phase shift the endogenous rhythm similar to light. The "clock genes" period1 (per1) and per2, which show circadian oscillation within the suprachiasmatic nucleus, have been attributed a role in light-induced resetting of the mammalian circadian clock due to rapid induction of the period (per) genes after light stimulation at night. Using a rat in vitro brain slice model, we demonstrate by quantitative in situ hybridization histochemistry that the diurnal alteration in expression of both per genes in the suprachiasmatic nucleus was retained in vitro. In the model, we examined the effects of pituitary adenylate cyclase-activating polypeptide and glutamate alone and in combination on per1 and per2 gene expression at late subjective night (circadian time 19). Glutamate administration (10(-3)M) induced both per1 and per2 gene expression in the suprachiasmatic nucleus of the brain slice within 1h. The per gene responses were similar to the induction of gene expression observed after light stimulation in vivo at late night. Pituitary adenylate cyclase-activating polypeptide (10(-6)M) administered alone had no effect on the per gene expression, but when pituitary adenylate cyclase-activating polypeptide in micromolar concentration was applied before glutamate, the neuropeptide blocked the glutamate-induced per1 and per2 gene expression in the suprachiasmatic nucleus. In contrast to the lack of effect of pituitary adenylate cyclase-activating polypeptide itself in micromolar concentration, pituitary adenylate cyclase-activating polypeptide (10(-9)M) induced both per1 and per2 gene expression, an effect which was not augmented by co-application of glutamate. Our results provide the molecular substrate for the previous electrophysiological findings that pituitary adenylate cyclase-activating polypeptide in high concentration is able to block glutamate-induced phase advance at late night, and that the peptide in low concentration can induce a phase advance similar to light and glutamate.

Estradiol down regulates expression of vasoactive intestinal polypeptide receptor type-1 in breast cancer cell lines.

Three breast carcinoma cell lines were tested for 17beta-estradiol (E(2)) mediated regulation of vasoactive intestinal polypeptide receptor type-1 (VPAC(1)) expression. In all three, E(2) was found to down-regulate the mRNA level. We studied T47D cells in more details and found a 25 and 70% decrease in the VPAC(1) mRNA level upon 7 and 48 h of E(2) treatment, respectively. The number of vasoactive intestinal polypeptide (VIP) binding sites was reduced 66% upon treatment with E(2) for 72 h. After cycloheximide pretreatment, the E(2) mediated mRNA reduction was attenuated from 50% to 25% after 24 h suggesting the effect to be at least partly independent of protein synthesis. Experiments with the transcriptional inhibitor actinomycin D showed that E(2) did not influence the VPAC(1) mRNA half-life while nuclear run-on experiments indicated that E(2) decreased the VPAC(1) transcription rate. Two antiestrogens: ICI 182780 (ICI) and 4-hydroxy-tamoxifen (4-OHT) mediated a concentration dependent inhibition of E(2)'s effect on the mRNA level. Transient transfection with reporter-gene constructs containing various portions of the VPAC(1) 5'-flanking sequence revealed the most proximal 100 bp to be essential for the basal transcriptional activity. However, E(2) did not influence the expression of the reporter gene using up to 3250 bp of the VPAC(1) 5'-flariking region.