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The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography–mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.

Early growth response transcription factor 1 (EGR1) is expressed in the suprachiasmatic nucleus (SCN) after light stimulation. We used EGR1-deficient mice to address the role of EGR1 in the clock function and light-induced resetting of the clock. The diurnal rhythms of expression of the clock genes BMAL1 and PER1 in the SCN were evaluated by semi-quantitative in situ hybridization. We found no difference in the expression of PER1 mRNA between wildtype and EGR1-deficient mice; however, the daily rhythm of BMAL1 mRNA was completely abolished in the EGR1-deficient mice. In addition, we evaluated the circadian running wheel activity, telemetric locomotor activity, and core body temperature of the mice. Loss of EGR1 neither altered light-induced phase shifts at subjective night nor affected negative masking. Overall, circadian light entrainment was found in EGR1-deficient mice but they displayed a reduced locomotor activity and an altered temperature regulation compared to wild type mice. When placed in running wheels, a subpopulation of EGR1-deficient mice displayed a more disrupted activity rhythm with no measurable endogenous period length (tau). In conclusion, the present study provides the first evidence that the circadian clock in the SCN is disturbed in mice deficient of EGR1.

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.

The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC1Rs as PACAP had >100-fold higher efficacy than VIP, and the PAC1R selective agonist Maxadilan potently induced PACAP gene expression. Experiments with pharmacological kinase inhibitors revealed that both PKA and novel but not conventional PKC isozymes were involved in the PACAP auto-regulation. Inhibition of MAPK/ERK kinase (MEK) also impeded the induction, and we found that PKA, novel PKC and ERK acted in parallel and were thus not part of the same pathways. The expression of the transcription factor EGR1 previously ascribed as target of PACAP signalling was found to be transiently induced by PACAP and pharmacological inhibition of either PKC or MEK1/2 abolished PACAP mediated EGR1 induction. In contrast, inhibition of PKA mediated increased PACAP mediated EGR1 induction. Experiments using siRNA against EGR1 to lower the expression did however not affect the PACAP auto-regulation indicating that this immediate early gene product is not part of PACAP auto-regulation in NB-1 cells. We here reveal that in NB-1 neuroblastoma cells, PACAP induces its own expression by activation of PAC1R, and that the signalling is different from the PAC1R signalling mediating induction of VIP in the same cells. PACAP auto-regulation depends on parallel activation of PKA, novel PKC isoforms, and ERK, while EGR1 does not seem to be part of the PACAP auto-regulation.

Neurons of the hypothalamic suprachiasmatic nucleus (SCN) express clock genes, which regulate their own transcription and generate daily output signals driving circadian rhythmic behavior and physiology. The neuropeptide VIP and its specific receptor, the VPAC2 receptor, are important for synchronization of clock neurons. In the present study, we characterized PER1 and PER2 expressing neurons in wild-type and VPAC2-deficient mice. We found evidence for distinct spatiotemporal circadian oscillation in the expression of the PER genes in two separate clusters of SCN neurons. In wild-type mice corresponding to the SCN shell and ventral core, high expression of PER was found at lights-off most likely representing an evening clock (E-clock). In another smaller cluster of neurons located in the central core of the SCN, PER expression peaks in antiphase at lights-on and could represent a morning clock (M-clock). BMAL1 immunoreactivity was found to be expressed in antiphase to PER in M and E neurons, respectively. PER was found in 98% of neurons expressing vasopressin (AVP) and in 92% of VIP neurons. The chemotype of M neurons was not identified. M but not E cells were responsive to long but not short photoperiods. The expression of the VPAC2 receptor was found in both M and E cells, and VPAC2-deficient mice displayed markedly blunted PER expression in both cell clusters of the SCN. Conclusion: These observations support the existence of M and E clocks involved in circadian and seasonal adaptation, which seem dependent on intact VIP/VPAC2 signaling in the SCN.

The intraocular pressure of mice displays a daily rhythmicity being highest during the dark period. The present study was performed to elucidate the role of the circadian clock and light in the diurnal and the circadian variations in intraocular pressure in mice, by using animals with disrupted clock function (VPAC2 receptor knockout mice) or impaired light information to the clock (PACAP knockout mice). In wildtype mice, intraocular pressure measured under light/dark conditions showed a statistically significant 24 h sinusoidal rhythm with nadir during the light phase and peak during the dark phase. After transfer of the wildtype mice into constant darkness, the intraocular pressure increased, but the rhythmic changes in intraocular pressure continued with a pattern identical to that obtained during the light/dark cycle. The intraocular pressure in VPAC2 receptor deficient mice during light/dark conditions also showed a sinusoidal pattern with significant changes as a function of a 24 h cycle. However, transfer of the VPAC2 receptor knockout mice into constant darkness completely abolished the rhythmic changes in intraocular pressure. The intraocular pressure in PACAP deficient mice oscillated significantly during both 24 h light and darkness and during constant darkness. During LD conditions, the amplitude of PACAP deficient was significantly lower compared to wildtype mice, resulting in higher daytime and lower nighttime values. In conclusion, by studying the VPAC2 receptor knockout mouse which lacks circadian control and the PACAP knockout mouse which displays impaired light signaling, we provided evidence that the daily intraocular pressure rhythms are primarily generated by the circadian master clock and to a lesser extent by environmental light and darkness.

Melanopsin is expressed in a subpopulation of retinal ganglion cells rendering these cells intrinsically photosensitive (ipRGCs). The ipRGCs are the primary RGCs mediating light entrainment of the circadian clock and control of the pupillary light reflex, light regulated melatonin secretion and negative masking behaviour. Previous studies have demonstrated that melanopsin expression in albino rats is regulated by light and darkness. The present study was undertaken to study the influence of light and darkness during the circadian day and after extended periods of constant light and darkness on melanopsin expression in the pigmented retina of the Brown Norwegian rat (Rattus norvegicus). The diurnal and circadian expressions were examined in retinal extracts from rats euthanized every 4 h during a 24 h light/dark (LD) and a 24 h dark cycle (DD) using quantitative real-time PCR and Western blotting. To study whether light regulates melanopsin expression, rats were sacrificed after being placed in either constant light (LL) or darkness for 3 or 21 d. Flat mount retinas from animals kept during either LL or DD were also examined by immunohistochemistry. Melanopsin mRNA expression displayed a significant rhythmic change during the LD cycle with peak expression around dusk and nadir at dawn. Melanopsin protein also changed over the LD cycle with peak expression at the end of the night and nadir at dusk. Rhythmic expression of melanopsin mRNA but not melanopsin protein was found in constant darkness. After 3 or 21 d in either LL or DD melanopsin mRNA expression was unaltered. Melanopsin protein was at the same high level after 3 and 21 d in DD, whereas a significant decrease was found after prolonging the light period for 3 or 21 d. The change in melanopsin protein was primarily due to change in immunoreactivity in the dendritic processes. In conclusion we found that light and darkness are important for regulation of melanopsin protein expression whereas input from a retinal networks regulates melanopsin mRNA expression. It is likely to speculate that altered level of melanopsin is one way in which the retina adapts to environmental light and darkness conditions ensuring optimal light sensitivity for the transmission to the brain.

Melanopsin retinal ganglion cells (mRGCs) are intrinsically photosensitive RGCs deputed to non-image forming functions of the eye such as synchronization of circadian rhythms to light-dark cycle. These cells are characterized by unique electrophysiological, anatomical and biochemical properties and are usually more resistant than conventional RGCs to different insults, such as axotomy and different paradigms of stress. We also demonstrated that these cells are relatively spared compared to conventional RGCs in mitochondrial optic neuropathies (Leber's hereditary optic neuropathy and Dominant Optic Atrophy). However, these cells are affected in other neurodegenerative conditions, such as glaucoma and Alzheimer's disease. We here review the current evidences that may underlie this dichotomy. We also present our unpublished data on cell experiments demonstrating that melanopsin itself does not explain the robustness of these cells and some preliminary data on immunohistochemical assessment of mitochondria in mRGCs.

The neuropeptide PACAP is expressed throughout the central and peripheral nervous system where it modulates diverse physiological functions including neuropeptide gene expression. We here report that in human neuroblastoma NB-1 cells PACAP transiently induces its own expression. Maximal PACAP mRNA expression was found after stimulation with PACAP for 3h. PACAP auto-regulation was found to be mediated by activation of PACAP specific PAC

The circadian time-keeping system consists of clocks in the suprachiasmatic nucleus (SCN) and in peripheral organs including an adrenal clock linked to the rhythmic corticosteroid production by regulating steroidogenic acute regulatory protein (StAR). Clock cells contain an autonomous molecular oscillator based on a group of clock genes and their protein products. Mice lacking the VPAC2 receptor display disrupted circadian rhythm of physiology and behaviour, and therefore, we using real-time RT-PCR quantified (1) the mRNAs for the clock genes Per1 and Bmal1 in the adrenal gland and SCN, (2) the adrenal Star mRNA and (3) the serum corticosterone concentration both during a light/dark (L/D) cycle and at constant darkness in wild type (WT) and VPAC2 receptor-deficient mice (VPAC2-KO). We also examined if PER1 and StAR were co-localised in the adrenal steroidogenic cells. Per1 and Bmal1 mRNA showed a 24-h rhythmic expression in the adrenal of WT mice under L/D and dark conditions. During a L/D cycle, the adrenal clock gene rhythm in VPAC2-KO mice was phase-advanced by approximately 6 h compared to WT mice and became arrhythmic in constant darkness. A significant 24-h rhythmic variation in the adrenal Star mRNA expression and circulating corticosterone concentration was similarly phase-advanced during the L/D cycle. The loss of adrenal clock gene rhythm in the VPAC2 receptor knockout mice after transfer into constant darkness was accompanied by disappearance of rhythmicity in Star mRNA expression and serum corticosterone concentration. Double immunohistochemistry showed that the PER1 protein and StAR were co-localised in the same steroidogenic cells. Circulating corticosterone plays a role in the circadian timing system and the misaligned corticosterone rhythm in the VPAC2 receptor knockout mice could be involved in their abnormal rhythms of physiology.

Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) have important local functions within the myocardium, where they protect against accelerated fibrosis. As circadian expression of cardiac natriuretic peptides could be of importance in local cardiac protection against disease, we examined the diurnal changes of the mRNAs encoding ANP, BNP, and their common receptor NPR-A in atrial and ventricular myocardium. Forty eight mice were killed at the following ZT times: 4, 8, 12, 16, 20, and 24, where ZT designates Zeitgeber; ZT 0 corresponds to lights ON and ZT 12 corresponds to lights OFF. Eight animals (4 males and 4 females) were included at each time point. Another 48 animals were killed during the second cycle of dark/dark (designated Circadian Time or CT: CT 4, CT 8, CT 12, CT 16, CT 20, and CT 24). The cellular contents of the clock genes Per1 and Bmal1 as well as ANP, BNP, and their common receptor (NPR-A) were determined using RT-PCR. Per1 and Bmal1 mRNA contents oscillated in antiphase in both atrial and ventricular regions, where Bmal1 mRNA peaked 12h out of phase relative to Per1 mRNA. ANP and NPR-A atrial mRNA contents revealed borderline significant diurnal changes, whereas ventricular BNP mRNA contents exhibited pronounced oscillation during constant darkness with nadir at CT 12 (P0.0001). In conclusion, we report a chamber-dependent circadian profile of cardiac BNP mRNA contents, which is not paralleled by the related ANP gene. Our findings suggest that the BNP mRNA pattern could be associated with increased cardiac susceptibility and response to disease.

J. Neurochem. (2010) 112, 797–806. Abstract The photopigment melanopsin is expressed in a subtype of mammalian ganglion cells in the retina that project to the circadian clock in the hypothalamic suprachiasmatic nucleus to mediate non-visual light information. Melanopsin renders these retinal ganglion cells intrinsically photosensitive and the cells respond to light by a membrane depolarization and induction of the immediate early response gene Fos. Previous studies showed that the light activated melanopsin-induced signaling, the phototransduction, leading to depolarization of the membrane resembles the invertebrate opsins, which involves a Gαq/11 coupled phospholipase C activation. However, the signaling proteins mediating melanopsin-induced Fos expression are unresolved. In this study, we examined the phototransduction leading to Fos expression in melanopsin-transfected PC12 cells. A pivotal role of the extracellular signal-regulated protein kinase 1/2 (ERK1/2) was found as pharmacological blockage of this kinase suppressed the light-induced Fos expression. Illumination increased the inositol phosphate turnover and induced phosphorylation of ERK1/2 and p38 but not the c-Jun N-terminal kinase. The Gαq/11 protein inhibitor YM254890 attenuated these intracellular light responses. Our data strongly indicate that Gαq/11-mediated ERK1/2 activation is essential for expression of Fos upon illumination of melanopsin-expressing PC12 cells.

Ganglia expressing the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) innervate vasoactive intestinal peptide (VIP) containing neurons suggesting a role of PACAP in regulating VIP expression. Human NB-1 neuroblastoma cells were applied to study PACAP regulated VIP gene expression aiming to identify the receptor and the signaling proteins involved. The PACAP receptor subtype PAC1 induced VIP gene expression as (i) PACAP and the PAC1 receptor agonist maxadilan were equally efficient and approximately 200-fold more potent than VIP, and (ii) PACAP6-38 and PG99-465, antagonists of PAC1 and VPAC2 receptors, respectively, abolished and did not affect the PACAP-induced VIP mRNA expression, respectively. A pivotal role of PKA was implicated in addition to partial involvement of PKC and ERK1/2 in PACAP-induced VIP gene expression as H-89, Bisindolylmaleimide I (BIS), Gö6976 and U0126 attenuated the VIP mRNA expression by 93%, 58%, 58% and 40%, respectively. PACAP modulated the phosphorylation of ERK1/2 (pERK1/2) and CREB/ATF-1 (pCREB/ATF-1) concomitant with a translocation of PKA to the nucleus. Inhibition of conventional PKC isoforms and MEK1/2 completely abolished pERK1/2 without affecting PACAP induced pCREB/ATF-1. In contrast, inhibiting PKA attenuated PACAP induced pCREB/ATF-1. PACAP also enhanced the FOS gene expression and individual presence of H-89, BIS, Gö6976 and U0126 partially attenuated the PACAP induced FOS mRNA expression. Combining the kinase inhibitors completely suppressed the PACAP induced FOS mRNA expression. Immunoblotting confirmed expression of FOS protein upon addition of PACAP, which was diminished by impairment of PKC, ERK1/2 and PKA activities. The resemblance of the signaling pathways involving concomitant activities of PKC, ERK1/2 and PKA in PACAP regulation of the FOS and VIP gene expressions suggest for the first time a role of FOS in PACAP-induced VIP gene expression in human NB-1 neuroblastoma cells.

Melanopsin is an opsin expressed in the plasma membrane of retinal ganglion cells that mainly project to the circadian clock and thus is important for nonvisual responses to light. Rat melanopsin contains two potential sites (Asn31 and Asn35) for N-linked glycosylation in the N-terminal extracellular part. To investigate if melanopsin is N-linked glycosylated and whether N-bound glycans influence the response of melanopsin to light as evidenced by Fos mRNA induction, we transfected PC12 cells to stably express rat wild-type melanopsin or mutant melanopsin lacking both N-linked glycosylation sites. Immunoblotting for membrane-bound melanopsin from the PC12 cells transfected to express wild-type melanopsin disclosed two immunoreactive bands of 62 and 49 kDa. Removal of N-linked glycosylation by tunicamycin or PNGase F changed the 62 kDa band to a 55 kDa band, while the 49 kDa band corresponding to the core melanopsin protein was unaffected. Likewise, mutation of the two extracellular N-linked glycosylation sites gave a melanopsin size comparable to that of PNGase F or tunicamycin treatment (55 kDa). Further in vitro O-linked deglycosylation of wild-type or mutant melanopsin with O-glycosidase and neuraminidase converted the 55 kDa band to a 49 kDa band. Finally, neither in vivo N-linked deglycosylation nor mutations of the two N-linked glycosylation sites significantly affected melanopsin function measured by Fos induction after light stimulation. In conclusion, we have shown that heterologously expressed rat melanopsin is both N-linked and O-linked glycosylated and that N-linked glycosylation is not crucial for the melanopsin response to light.

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) mediates its physiological functions through activation of PAC1, VPAC1 and VPAC2 receptors, and the ubiquitous Ca 2+ -sensor calmodulin has been implicated in PACAP-induced signaling. The immediate early response gene FOS is a well-known marker of neuronal activation, so we used a human neuroblastoma cell line NB-1 to explore the role of calmodulin in PACAP-induced FOS gene expression. We observed both short-term and prolonged altered PACAP-mediated activation of the FOS gene in the presence of the calmodulin-antagonist W-7. NB-1 cells were shown to express PAC1 and VPAC2 receptors, and immunoprecipitation of both receptors displayed a co-association with calmodulin in the absence of Ca 2+ . Our findings indicate a novel mechanism of calmodulin in regulating PACAP signaling by possible interaction with the inactive state of PAC1 and VPAC2 receptors.

To examine for circadian rhythmicity, the messenger RNA (mRNA) amount of the clock genes Per1 and Per2 was measured in undifferentiated and nerve-growth-factor-differentiated PC12 cells harvested every fourth hour. Serum shock was needed to induce circadian oscillations, which in undifferentiated PC12 cultures lasted only one 24-h period, while in differentiated cultures, the rhythms continued for at least 3 days. Thus, neuronal differentiation provided PC12 cells the ability to maintain rhythmicity for an extended period. Both vasoactive intestinal polypeptide (VIP) and its receptor VPAC2 are expressed in the suprachiasmatic nucleus (SCN), and in agreement with VIP signaling being crucial for maintenance of rhythmicity, we found both VIP and VPAC2 mRNA increased after differentiation of PC12 cells. Pituitary adenylate cyclase activating polypeptide (PACAP) exerts time- and concentration-dependent effects on Per gene expression in the SCN. We added 1 nM and 1 microM PACAP to oscillating PC12 cells at times corresponding to midday and early and late night to evaluate whether the effects were similar as in SCN. Induction of Per1 mRNA was found at all three times, which differs from results in SCN. Thus, PC12 cells seem more useful for studying mechanisms behind acquirement of rhythmicity of cell cultures than for resetting of circadian rhythm.

Huntington's disease (HD) is a fatal genetic neurodegenerative disorder caused by a CAG triplet repeat expansion in the gene encoding the protein huntingtin. The most studied model of HD, the R6/2 transgenic mouse, replicates many features of the disease. In addition to motor, cognitive, and endocrine dysfunctions, these mice exhibit a progressive disruption of circadian rhythms. This is accompanied by an altered expression of the circadian clock genes in the suprachiasmatic nucleus/nuclei (SCN), the principal circadian pacemaker in the brain. The neuropeptide vasoactive intestinal polypeptide (VIP) and its receptor VPAC2 are highly expressed in the SCN, and VIPergic signaling plays an essential role in maintenance of ongoing circadian rhythmicity. We found a marked reduction in both VIP mRNA and VPAC2 receptor mRNA, quantified by RT-PCR, as well as a decrease in VIP immunostaining in the SCN of R6/2 mice. These changes were coupled to a disruption of circadian rhythm. We observed no loss of neurons in the SCN and therefore suggest that the changes in VIP and VPAC2 receptor are due to their decreased expression. In conclusion, we propose that impaired VIPergic signaling is an additional candidate mechanism for disruption of circadian rhythms in R6/2 mice.

PACAP in the retinohypothalamic tract mediates photic information to the suprachiasmatic nucleus via the PAC1 receptor. The diurnal and circadian VIP mRNA expressions in the suprachiasmatic nucleus of PAC1-/- and wild type mice were quantified. During light/dark cycles identical VIP mRNA rhythms were found while the oscillation pattern differed between the two types of animals during constant darkness. The results show that the circadian VIP mRNA expression is influenced by the absence of PAC1 signalling.

Intrinsically photosensitive retinal ganglion cells employ the photopigment melanopsin and provide light information to brain areas responsible for the regulation of circadian rhythms. The expression of melanopsin is regulated by environmental illumination, but it remains to be clarified whether the rods and cones are involved. Here, we examined the influence of 5 days of constant light and dark conditions on melanopsin mRNA and protein expression in newborn albino rats, in which functional rods and cones have not yet been developed. We found that the melanopsin mRNA level was unaffected, whereas the melanopsin protein level was more than two-fold higher in the darkness-adapted group than in pups raised in constant light. In pups raised during 12 : 12 h light/dark cycles, the melanopsin protein level was significantly higher during the day than at night. Our findings indicate that melanopsin protein changes are independent of input from the rods and cones.

The breast carcinoma cell line T47D was tested for 17 beta-estradiol (E2) mediated regulation of vasoactive intestinal polypeptide receptor type-1 (VPAC1) expression. E2 was found to downregulate the mRNA level. The number of VIP binding sites was reduced 66% on treatment with E2 for 72 h. Experiments with cycloheximide suggested that the effect was independent (at least partly so) of protein synthesis. Experiments with the transcriptional inhibitor, actinomycin D, showed that E2 did not influence the VPAC1 mRNA halflife. Both of two antiestrogens, ICI 182,780 and 4-hydroxy-tamoxifen, mediated a concentration dependent inhibition of the effect of E2 on the mRNA level. Transient transfection with reporter-gene constructs containing various portions of the VPAC1 5'-flanking sequence revealed the most proximal 100 bp to be essential for the basal transcriptional activity. However, E2 did not influence the expression of the reporter gene using up to 3,250 bp of the VPAC1 5'-flanking region.

The suprachiasmatic nucleus (SCN) generates circadian rhythms which are synchronised to the environmental light/dark cycle via the retinohypothalamic tract (RHT). Pituitary adenylate cyclase activating polypeptide (PACAP) and glutamate, two transmitters co-stored in the rat retinohypothalamic tract, are involved in photic entrainment of the circadian pacemaker, but their functional interplay is poorly understood. Homer proteins are involved in glutamatergic receptor function and signalling. By quantitative in situ hybridisation histochemistry we found that light stimulation of rats at early and late night induced Homer-1 gene expression in the SCN at time points where light induces phase-delay or phase-advance, respectively. Using a rat brain slice model Homer-1 mRNA levels in the SCN displayed a modest diurnal variation similar to that in vivo. The changes in Homer-1 gene expression after in vitro stimulation with PACAP and/or glutamate differed at early and late night. Nanomolar PACAP induced Homer-1 gene expression at both early and late night while glutamate was only able to increase Homer-1 mRNA level at early night. PACAP in micromolar concentration had no effect per se, but inhibited the glutamate induced Homer-1 response at early night, while at late night co-administration of PACAP and glutamate mediated a slight induction of Homer-1 gene expression. In conclusion, the RHT transmitters PACAP and glutamate could be responsible for the light-induced expression of Homer-1 in the SCN, and Homer-1 seems to be differentially regulated by the two transmitters at early and late night.

We established a cell line (HEK-hMel) expressing melanopsin in a tetracycline dependent manner to elucidate new aspects of melanopsin's light response. Different light stimuli were evaluated using FOS expression as response parameter. Immunoblotting was used to evaluate expression of melanopsin and FOS and qPCR to quantify FOS mRNA responses. The magnitude of the FOS response was found to correlate with the amount of melanopsin expressed by the cells, and a transient FOS mRNA induction followed by FOS protein still elevated after 24 h of illumination was revealed. Exposing the cells to darkness after light resulted in reduction of the response compared to exposure to light solely showing dependency on continuous light. Increasing irradiances of blue light (480 nm) up to 10(11) quanta cm(-2) s(-1) elicited steep increases in FOS mRNA, while increases between 10(12) and 5 × 10(13) quanta cm(-2) s(-1) resulted in equally high FOS expression. The HEK-hMel cells were used to characterize facets of melanopsin's light-induced FOS response not approachable in vivo. Novel information such as dependency of the FOS response on both melanopsin amount and light intensity in addition to a detailed time-course of both FOS mRNA and protein were revealed.

The concentration of pituitary adenylyl cyclase-activating polypeptide [PACAP-(1–38)] in porcine adrenal glands amounted to 14 ± 3 pmol/g tissue. PACAP immunoreactive (PACAP-IR) fibers innervated adrenal chromaffin cells (often co-localized with choline acetyltransferase). Subcapsular fibers traversed the cortex-innervating endocrine cells and blood vessels [some co-storing mainly calcitonin gene-related peptide but also vasoactive intestinal polypeptide (VIP)]. PACAP-IR fibers were demonstrated in the splanchnic nerves, whereas IR adrenal nerve cell bodies were absent. In isolated, vascularly perfused adrenal gland, splanchnic nerve stimulation (16 Hz) and capsaicin (10−5 M) increased PACAP-(1–38) release (1.6-fold and 6-fold respectively, P = 0.02). PACAP-(1–38) dose-dependently stimulated cortisol (2 × 10−10 M; 24-fold increase, P = 0.02) and chromogranin A fragment (2 × 10−9 M; 15-fold increase, P = 0.05) secretion. Both were strongly inhibited by the PAC1/VPAC2 receptor antagonist PACAP-(6–38) (10−7 M). PACAP-(6–38) also inhibited splanchnic nerve (10 Hz)-induced cortisol secretion but lacked any effect on splanchnic nerve-induced pancreastatin secretion. PACAP-(1–38) (2 × 10−10 M) decreased vascular resistance from 5.5 ± 0.6 to 4.6 ± 0.4 mmHg · min · ml−1. PACAP-(6–38) had no effect on this response. We conclude that PACAP-(1–38) may play a role in splanchnic nerve-induced adrenal secretion and in afferent reflex pathways.

In many ganglia, the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) innervates nerve cell bodies containing the homologous neuropeptide vasoactive intestinal polypeptide (VIP). We therefore investigated whether PACAP affected the VIP gene expression and elucidated the molecular mechanisms using the human neuroblastoma cell line NB-1. A concentration dependent induction of the VIP mRNA level was found upon PACAP stimulation. Five nM PACAP mediated transient elevation of the VIP mRNA being evident after 2 h, the maximal 65-fold induction was reached after 6-8 h and hereafter the level decreased rapidly. In cell extracts, the concentration of immunoreactive VIP was elevated four-fold upon PACAP stimulation for 8 h, and it remained elevated during the next 40 h. In conditioned medium, a stable 20-fold VIP increase was seen after 8-24 h. Experiments with the translational inhibitor cycloheximide showed a direct effect of PACAP on the VIP mRNA level, and nuclear run-on assays revealed a three- to four-fold enhancement of the VIP gene transcription rate after PACAP stimulation. The VIP mRNA induction was abolished by transcriptional inhibition with the actinomycin D, and PACAP did not seem to mediate any changes in the VIP mRNA half-life. However, the VIP mRNA level seemed very stable during the transcriptional cessation. Reporter gene constructs were used to evaluate involvement of the VIP CRE site in the PACAP mediated induction of the VIP gene transcription. Mutation of the CRE site did not abolish the induction suggesting it to be of minor if any importance for the induction. In conclusion, the PACAP mediated induction of the VIP gene expression suggests that PACAP released from nerve terminals could influence the function of VIP'ergic neurons in target tissues.

Background: Melanopsin is a retinal light-sensitive receptor involved in non-image forming functions including circadian timing. Results: The intracellular C-terminal part of melanopsin was shown to be phosphorylated at distinct functional sites in response to light and dark. Conclusion: Phosphorylation status of melanopsin influences its functions. Significance: Distinct sites of phosphorylation are important in determining the signaling properties of melanopsin., The G protein-coupled light-sensitive receptor melanopsin is involved in non-image-forming light responses including circadian timing. The predicted secondary structure of melanopsin indicates a long cytoplasmic tail with many potential phosphorylation sites. Using bioinformatics, we identified a number of amino acids with a high probability of being phosphorylated. We generated antibodies against melanopsin phosphorylated at Ser-381 and Ser-398, respectively. The antibody specificity was verified by immunoblotting and immunohistochemical staining of HEK-293 cells expressing rat melanopsin mutated in Ser-381 or Ser-398. Using the antibody recognizing phospho-Ser-381 melanopsin, we demonstrated by immunoblotting and immunohistochemical staining in HEK-293 cells expressing rat melanopsin that the receptor is phosphorylated in this position during the dark and dephosphorylated when light is turned on. On the contrary, we found that melanopsin at Ser-398 was unphosphorylated in the dark and became phosphorylated after light stimulation. The light-induced changes in phosphorylation at both Ser-381 and Ser-398 were rapid and lasted throughout the 4-h experimental period. Furthermore, phosphorylation at Ser-381 and Ser-398 was independent of each other. The changes in phosphorylation were confirmed in vivo by immunohistochemical staining of rat retinas during light and dark. We further demonstrated that mutation of Ser-381 and Ser-398 in melanopsin-expressing HEK-293 cells affected the light-induced Ca2+ response, which was significantly reduced as compared with wild type. Examining the light-evoked Ca2+ response in a melanopsin Ser-381 plus Ser-398 double mutant provided evidence that the phosphorylation events were independent.

Pituitary adenylate cyclase activating polypeptide (PACAP), a potent stimulator of cAMP formation, has a widespread distribution in neuronal elements of the central nervous system and a number of peripheral organs. Recently, PACAP has also been shown to be stage specifically expressed in the spermatogenic cells from the rat testis. This prompted us to examine the cell-specific expression of PACAP during the estrous cycle in the rat ovary by in situ hybridization with a digoxiginin UTP-labeled RNA probe and by immunohistochemistry using a specific monoclonal antibody. Ovaries from PMS gonadotropin (PMSG)/human CG (hCG) treated immature animals were examined 0, 12, 24, 48, 54, 57, 58.5, 60, 61.5, 63, 66, 72, and 96 h after PMSG treatment. Ovaries from adult cyclic rats were examined at 1000 h on each day of the estrous cycle and at 0400 h on estrus morning. PACAP positive cells were observed in the preovulatory and ovulatory period, i.e. 6-18 h following hCG injection in the PMSG induced cycle and at 0400 h and 1000 h on estrus morning in adult cyclic animals. Both PACAP messenger RNA (mRNA) and PACAP immunoreactivity were observed in the majority of granulosa and cumulus cells from large preovulatory follicles, in the majority of the cells comprising the interstitial glandular tissue and in solitary theca cells of growing and mature follicles. In PMSG/hCG treated animals, positive signals were also observed in solitary theca and interstitial cells 12 h after PMSG injection. PACAP immunoreactive nerve fibers were observed in all ovaries examined, located in the interstitial glandular tissue or in the loose connective tissue. Northern blotting of ovarian tissue confirmed the transient preovulatory and ovulatory expression of PACAP. Three transcripts were observed. The most predominant band had a size of approximately 1.2 kilobase pairs, corresponding to the transcript observed in the testis. Two bands of 2.4 and 3.0 kilobase pairs, corresponding to the transcripts from neural tissue, were also observed. The complex distribution of PACAP positive cells and nerve fibers suggests that the peptide could be a local regulator of a number of the events that take place in the periovulatory period as well as during early folliculogenesis in the ovary.

Daily oscillations of clock genes have recently been demonstrated in the ovaries of several species. Clock gene knockout or mutant mice demonstrate a variety of reproductive defects. Accumulating evidence suggests that these rhythms act to synchronise the expression of specific ovarian genes to hypothalamo-pituitary signals and that they are regulated by one or both of the gonadotropins. The aim of this study has been to examine the spatio-temporal expression of the clock genes Per1 and Bmal1 during gonadotropin-independent and gonadotropin-dependent follicle development in the rat ovary. We have examined the ovaries of prepubertal rats, of prepubertal rats stimulated with equine chorionic gonadotropin (eCG)/human chorionic gonadotropin (hCG) and of hypophysectomised adult animals. Using quantitative reverse transcription with the polymerase chain reaction, in situ hybridisation histochemistry and immunohistochemistry, we have demonstrated that the expression of the two clock genes is low and arrhythmic in ovarian cells during early gonadotropin-independent follicle development in prepubertal animals and in hypophysectomised animals. We have also demonstrated that the expression of the clock genes becomes rhythmic following eCG stimulation in the theca interna cells and the secondary interstitial cells and that, following additional hCG stimulation, the expression of the clock genes also becomes rhythmic in the granulosa cells of preovulatory follicles. These findings link the initiation of clock gene rhythms in the rat ovary to the luteinising hormone receptor and suggest a functional link to androgen and progesterone production. In hypophysectomised animals, rhythmic clock gene expression is also observed in the corpora lutea and in secondary interstitial cells demonstrating that, in these compartments, entrainment of clock gene rhythms is gonadotropin-independent.

Neuroglobin (Ngb), a neuron-specific oxygen-binding globin with an unknown function, has been proposed to play a key role in neuronal survival. We have previously shown Ngb to be highly expressed in the rat suprachiasmatic nucleus (SCN). The present study addresses the effect of Ngb deficiency on circadian behavior. Ngb-deficient and wild-type (wt) mice were placed in running wheels and their activity rhythms, endogenous period and response to light stimuli were investigated. The effect of Ngb deficiency on the expression of Period1 (Per1) and the immediate early gene Fos was determined after light stimulation at night and the neurochemical phenotype of Ngb expressing neurons in wt mice was characterized. Loss of Ngb function had no effect on overall circadian entrainment, but resulted in a significantly larger phase delay of circadian rhythm upon light stimulation at early night. A light-induced increase in Per1, but not Fos, gene expression was observed in Ngb-deficient mice. Ngb expressing neurons which co-stored Gastrin Releasing Peptide (GRP) and were innervated from the eye and the geniculo-hypothalamic tract expressed FOS after light stimulation. No PER1 expression was observed in Ngb-positive neurons. The present study demonstrates for the first time that the genetic elimination of Ngb does not affect core clock function but evokes an increased behavioural response to light concomitant with increased Per1 gene expression in the SCN at early night.

Prepro-vasoactive intestinal peptide (preproVIP) was expressed in NIH 3T3 cells, and the preproVIP-derived peptides produced by the cells were analyzed by chromatography combined with sequence-specific radio-immunoanalysis. In accordance with what has previously been reported on processing in non-endocrine cell lines, the VIP precursor was processed poorly in these non-endocrine cells. Mainly an extended form of VIP could be detected in the media from the cells, and no immunoreactivity specific for amidated VIP was found. However, by changing the dibasic cleavage site positioned N-terminal to the VIP sequence in the precursor into the consensus sequence (Arg,X,Lys/Arg,Arg) for the ubiquitous processing enzyme furin, thought to process, e.g. insulin receptors, factor VII, and by deleting residues 156–170 in the VIP precursor, expression of amidated VIP was obtained in this fibroblast cell line. Peptides from the wild-type VIP precursor as well as peptides from the mutated VIP precursor were found to be able to stimulate the adenylate cyclase in cells expressing the VIP receptor.

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, Per 1, 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.

Circadian rhythms are generated by endogenous clocks in the central brain oscillator, the suprachiasmatic nucleus, and peripheral tissues. The molecular basis for the circadian clock consists of a number of genes and proteins that form transcriptional/translational feedback loops. In the mammalian gonads, clock genes have been reported in the testes, but the expression pattern is developmental rather than circadian. Here we investigated the daily expression of the two core clock genes, Per1 and Per2, in the rat ovary using real-time RT-PCR, in situ hybridization histochemistry, and immunohistochemistry. Both Per1 and Per2 mRNA displayed a statistically significant rhythmic oscillation in the ovary with a period of 24 h in: 1) a group of rats during proestrus and estrus under 12-h light,12-h dark cycles; 2) a second group of rats representing a mixture of all 4 d of the estrous cycle under 12-h light,12-h dark conditions; and 3) a third group of rats representing a mixture of all 4 d of estrous cycle during continuous darkness. Per1 mRNA was low at Zeitgeber time 0–2 and peaked at Zeitgeber time 12–14, whereas Per2 mRNA was delayed by approximately 4 h relative to Per1. By in situ hybridization histochemistry, Per mRNAs were localized to steroidogenic cells in preantral, antral, and preovulatory follicles; corpora lutea; and interstitial glandular tissue. With newly developed antisera, we substantiated the expression of Per1 and Per2 in these cells by single/double immunohistochemistry. Furthermore, we visualized the temporal intracellular movements of PER1 and PER2 proteins. These findings suggest the existence of an ovarian circadian clock, which may play a role both locally and in the hypothalamo-pituitary-ovarian axis.

Circadian rhythms are daily adjusted to the environmental day/night cycle by photic input via the retinohypothalamic tract (RHT). Recent studies indicate that melanopsin, a newly identified opsin-like molecule, is involved in the light responsiveness of retinal ganglion cells (RGCs) constituting the RHT. In the present study, we examined the expression of melanopsin at the mRNA and protein level during a day/night cycle and during prolonged periods of light and darkness in the retina of albino Wistar rats. We observed a diurnal change in melanopsin, with mRNA level being highest at early subjective night and protein level highest at late subjective day. Prolonged exposure to darkness significantly increased melanopsin mRNA level as early as the first day, and the expression continued to increase during 5 d in darkness. The decrease in mRNA level during exposure to constant light was slower. After 48 h of light, the melanopsin mRNA level was significantly reduced, and an almost undetectable level was found after 5 d. The induction of melanopsin by darkness was even more pronounced if darkness was preceded by light suppression for 5 d. By use of immunohistochemistry, we showed that darkness increased the amount of protein in the dendritic processes, resulting in a dense network covering the entire retina. Constant light decreased melanopsin immunostaining time dependently, beginning in the distal dendrites and progressing to the proximal dendrites and the soma. Our observations suggest that the intrinsic light-responsive RGCs adapt their expression of the putative circadian photopigment melanopsin to environmental light and darkness.

PURPOSE. The putative circadian photoreceptor melanopsin is found in rodents in a subpopulation of intrinsic light-sensitive retinal ganglion cells (RGCs) constituting the retinohypothalamic tract (RHT). The study was conducted to determine whether melanopsin is expressed in the human retina and costored with the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP), a marker for the RHT, projecting to the suprachiasmatic nucleus (SCN). Furthermore, whether melanopsin expression is conserved in retinas of blind patients with severe retinal degeneration was investigated. METHODS. In situ hybridization and immunohistochemistry was used to demonstrate melanopsin synthesis in human eyes of 17 donors and two postmortem hypothalami containing the SCN. The coexistence of melanopsin and PACAP in elements of the retinohypothalamic tract was studied by dual-labeling immunocytochemistry. RESULTS. Melanopsin expression was found in a subpopulation of RGCs located in the ganglion cell layer and displaced in the inner nuclear cell layer. Melanopsin-containing cells comprised approximately 0. 8% of all RGCs, with a distinct morphology characterized by two to four dendritic processes constituting a panretinal network. Melanopsin immunoreactivity was primary present at perikaryal boundaries and neuronal processes and to some extent also in the cytoplasm. PACAP and melanopsin were colocalized in the RGCs and PACAP-containing nerve fibers, seemingly innervating the retinorecipient part of the SCN. Melanopsin-expressing RGCs were conserved in retinas of blind patients with severe degeneration of the outer and/or inner layers. CONCLUSIONS. Given the expression of melanopsin in PACAPcontaining RGCs of the human RHT, this photoreceptor is a likely first base in the chain of events leading to photoentrainment of both normal and blind people. (Invest Ophthalmol Vis Sci. 2004;45:4202‐4209) DOI:10.1167/iovs.04-0313

Mammalian circadian rhythms generated in the hypothalamic suprachiasmatic nuclei are entrained to the environmental light/dark cycle via a monosynaptic pathway, the retinohypothalamic tract (RHT). We have shown previously that retinal ganglion cells containing pituitary adenylate cyclase-activating polypeptide (PACAP) constitute the RHT. Light activates the RHT via unknown photoreceptors different from the classical photoreceptors located in the outer retina. Two types of photopigments, melanopsin and the cryptochromes (CRY1 and CRY2), both of which are located in the inner retina, have been suggested as "circadian photopigments." In the present study, we cloned rat melanopsin photopigment cDNA and produced a specific melanopsin antibody. Using in situ hybridization histochemistry combined with immunohistochemistry, we demonstrate that the distribution of melanopsin was identical to that of the PACAP-containing retinal ganglion cells. Colocalization studies using the specific melanopsin antibody and/or cRNA probes in combination with PACAP immunostaining revealed that melanopsin was found exclusively in the PACAP-containing retinal ganglion cells located at the surface of somata and dendrites. These data, in conjunction with published action spectra analyses and work in retinally degenerated (rd/rd/cl) mutant mice, suggest that melanopsin is a circadian photopigment located in retinal ganglion cells projecting to the biological clock.

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.

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.

The two structurally related peptides, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP), are present in cerebral vascular nerve fibers. Biologic actions of VIP are exerted through two receptors, VPAC1 and VPAC2, having similar binding affinity for both VIP and PACAP. In the current study, the authors have developed a specific antibody against the rVPAC1 receptor to examine the localization of rVPAC1 immunoreactivity in cerebral arteries and arterioles of the rat by immunohistochemistry using fluorescence confocal microscopy. Specificity of the antiserum was ensured by immunoblotting and immunocytochemistry of cells transfected with cDNA encoding the different PACAP-VIP receptor subtypes. The rVPAC1 receptor immunoreactivity was localized to the plasmalemma of circularly orientated smooth muscle cells on superficial cerebral arteries and arterioles taken from the basal surface of the brain. By double immunostaining VIP immunoreactive nerve fibers and, to a lesser extent, those containing PACAP were shown to have intimate contact with the receptor protein. Vasoactive intestinal polypeptide and PACAP containing cerebrovascular nerve fibers were found in separate nerve populations with different distribution pattern and density. In brain sections processes of cortical VIP-, but not PACAP-, containing neurons seemed to innervate the rVPAC1 receptor of pial arterioles on the brain surface. The current findings provide the neuroanatomical substrate for a role of VIP and maybe PACAP in the regulation of cerebral blood flow.

The effects of carbamylcholine (CCh) on the gene expression of the neuropeptide vasoactive intestinal polypeptide (VIP) were studied using two human neuroblastoma cell lines, NB-1 and BE(2)M17. CCh caused a fast increase in VIP mRNA level in both cell lines which was followed by an increase in VIP immunoreactivity. The time-course of the induction of both mRNA and peptide differed, however, between the two cell lines. No morphological changes of the cells were observed during 6 days of stimulation. The effect was mediated by the muscarinic class of acetylcholine receptors, since it could be totally abolished by atropine. Since CCh caused an accumulation of inositol-1,4,5-triphosphate, it is likely that muscarinic receptor subtype M1, M3 or M5 is involved. Experiments with the translational inhibitor, cycloheximide, showed that CCh mediated a direct effect on the VIP gene expression. By combining gel permeation chromatography with radioimmunoassays using antisera specific for the various VIP-precursor products, immunoreactive peaks eluting as the synthetic peptides were found in both cell lines. In addition, earlier eluting peaks which could represent partially processed or extended VIP forms were found. After CCh induction the concentration of all preproVIP-derived products increased, and there was a tendency towards a shift to more fully processed VIP. The findings give new evidence for a direct regulation of VIP gene expression in human neuronal cells by cholinergic agents.

We report here data on the expression of the various sequences derived from the prepro-vasoactive intestinal polypeptide (VIP) precursor and VIP mRNA in the anterior pituitary gland and cerebral cortex of hypothyroid and hyperthyroid rats. Using specific antisera to each of the prepro-VIP sequences we demonstrated an increase of all prepro-VIP derived sequences, and accordingly, found that the number of cells expressing each of these sequences were markedly augmented in the anterior pituitary of the hypothyroid rats. This was accompanied by a marked increase in VIP mRNA. In the cerebral cortex of the hypothyroid rats no changes were observed. In the pituitary of hyperthyroid animals a significant decrease was seen for prepro-VIP 22–79, VIP and prepro-VIP 156–170, whereas in the cerebral cortex a significant increase was observed for prepro-VIP 22–79, PHI and VIP. We were not able to demonstrate any changes in VIP mRNA in the cerebral cortex or pituitary of the hyperthyroid rats. Gel permeation chromatography and reverse-phase HPLC of extracts from cerebral cortex showed elution profiles identical to the synthetic counterparts. The reported data provide further evidence of a tissue-specific expression and regulation of the VIP gene products.

The expression of VIP precursor products: prepro-VIP(22-79), peptide histidine isoleucine (PHI), peptide histidine valine (PHV), prepro-VIP(111-122), VIP, prepro-VIP(156-170), and prepro-VIP mRNA in the anterior pituitary of estrogen-treated, ovariectomized rats, of ovariectomized controls, and of sham-operated controls was examined. Using radioimmunoassays based on antisera against each of the prepro-VIP sequences, we found that all sequences were expressed and markedly induced by estrogen, except PHI and PHV, which both were undetectable. By immunohistochemistry, it appeared that the number of cells immunoreactive for each of these sequences was increased in the estrogen-treated animals. However, PHI/PHV-immunoreactive cells could not be detected, despite the use of four different PHI antisera with different specificities. Estrogen treatment increased the prepro-VIP mRNA as judged by Northern blotting. In situ hybridization signals for both VIP mRNA and PHI mRNA were observed in few pituitary cells from control animals whereas strong positive signals were observed in a larger number of cells after estrogen treatment. The findings show that estrogen causes activation of the VIP gene expression in anterior pituitary cells, and that the absence of PHI and PHV probably is due to translational or posttranslational events.

The neuropeptide vasoactive intestinal polypeptide (VIP) has a broad range of functions, and its expression has been correlated with neuronal differentiation. Here we present data on the effects of retinoic acid (RA), a known modulator of neuronal differentiation, on VIP gene expression in the human neuroblastoma cell line NB-1. Morphological data, surprisingly, indicate that these cells are not differentiated concomitant with the increase in VIP gene expression. RA was found to exert a concentration-dependent induction of peptides derived from the VIP precursor molecule, prepro-VIP. The effect at both the messenger RNA (mRNA) level, evaluated by Northern blots, and the peptide level, measured by RIAs, was found to be slow and long lasting. No changes in the processing of prepro-VIP were observed using gel chromatography and RIAs specific for various prepro-VIP sequences. Also, the expression of mRNA for the prohormone-processing enzyme PC2, present in these cells, was not altered by RA. The lag period preceding the increase in VIP mRNA led to experiments with the translational inhibitor cycloheximide showing an indirect effect of RA on VIP mRNA expression. Northern blots revealed that at least three mRNAs encoding RA receptor were expressed and rapidly induced by RA in the cells, thus making them possible candidates for the intermediate protein(s) required from the induction of VIP gene expression.

We have studied the effect of the adenylate cyclase-stimulating agent forskolin on expression of components of the plasminogen activation system in the human fibrosarcoma cell line HT-1080. By enzyme-linked immunosorbent assays, forskolin was found to cause a 2 to 4-fold decrease in intracellular and culture medium levels of type-1 inhibitor of plasminogen activators (PAI-1) and tissue-type plasminogen activator (t-PA). This was true for cells not treated with other agents and for cells, in which the PAI-1 and t-PA levels had been increased 5 to 10-fold by treatment with dexamethasone. This down-regulation could be traced back to corresponding decreases in the cellular levels of PAI-1 and t-PA mRNAs. Of the two PAI-1 mRNAs, the 2.4 kb species was 5-fold decreased by forskolin in cells treated with dexamethasone, while the 3.4 kb transcript was unaffected; in cells not treated with dexamethasone, forskolin affected the two PAI-1 transcripts in parallel. These studies show that in addition to the many inducers of PAI-1, PAI-1 gene expression is also subject to negative modulation by cyclic AMP. They also show that t-PA gene expression, in contrast to the induction by cyclic AMP observed in many other cell lines, may also be subject to negative regulation by cyclic AMP. Thus, hormonal agents acting with cyclic AMP as a second messenger may be involved in down-regulating PAI-1 and t-PA expression in vivo. © 1990.

Etude de la regulation hormonale des enzymes appartenant a la famille de serine protease inhibiteur (plasminogen activator et urokinase)