Akikazu Mochiduki

Identification of autotaxin as a neurite retraction-inducing factor of PC12 cells in cerebrospinal fluid and its possible sources

Cerebrospinal fluid (CSF) induced neurite retraction of differentiated PC12 cells; the action was observed in 15 min (a rapid response) and the activity further increased until 6 h (a long‐acting response) during exposure of CSF to the cells. The CSF action was sensitive to monoglyceride lipase and diminished by homologous desensitization with lysophosphatidic acid (LPA) and by pretreatment with an LPA receptor antagonist Ki16425. Although fresh CSF contains LPA to some extent, the LPA content in the medium was increased during culture of PC12 cells with CSF. The rapid response was mimicked by exogenous LPA, and a long‐acting response was duplicated by a recombinant autotaxin, lysophospholipase D (lyso‐PLD). Although the lyso‐PLD substrate lysophosphatidylcholine (LPC) was not detected in CSF, lyso‐PLD activity and an ∼120‐kDa autotaxin protein were detected in CSF. On the other hand, LPC but not lyso‐PLD activity was detected in the conditioned medium of a PC12 cell culture without CSF. Among neural cells examined, leptomeningeal cells expressed the highest lyso‐PLD activity and autotaxin protein. These results suggest that leptomeningeal cells may work as one of the sources for autotaxin, which may play a critical role in LPA production and thereby regulate axonal and neurite morphological change.

Prolactin-Releasing Peptide Regulates the Cardiovascular System Via Corticotrophin-Releasing Hormone

Prolactin‐releasing peptide (PrRP)‐producing neurones are known to be localised mainly in the medulla oblongata and to act as a stress mediator in the central nervous system. In addition, central administration of PrRP elevates the arterial pressure and heart rate. However, the neuronal pathway of the cardiovascular effects of PrRP has not been revealed. In the present study, we demonstrate that PrRP‐immunoreactive neurones projected to the locus coeruleus (LC) and the paraventricular nucleus (PVN) of the hypothalamus. The c‐fos positive neurones among the noradrenaline cells in the LC, and the parvo‐ and magnocellular neurones in the PVN, were increased after central administration of PrRP. The arterial pressure and heart rate were both elevated after i.c.v. administration of PrRP. Previous studies have demonstrated that PrRP stimulated the neurones in the PVN [i.e. oxytocin‐, vasopressin‐ and corticotrophin‐releasing hormone (CRH)‐producing neurones], which suggests that PrRP may induce its cardiovascular effect via arginine vasopressin (AVP) or CRH. Although the elevation of blood pressure and heart rate elicited by PrRP administration were not inhibited by an AVP antagonist, they were completely suppressed by treatment with a CRH antagonist. Thus, we conclude that PrRP stimulated CRH neurones in the PVN and that CRH might regulate the cardiovascular system via the sympathetic nervous system.

Circadian transcriptional factor DBP regulates expression of Kiss1 in the anteroventral periventricular nucleus

The expression of Kiss1 in the anteroventral periventricular nucleus (AVPV) and its product, metastin/kisspeptin, show a circadian pattern with a peak in the evening, which shows a strong phase relationship with the time of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) surge in rodents. Here we report that a circadian transcriptional factor, albumin D-site binding protein (Dbp), was able to trigger mKiss1 transcription via the D-box, and this effect was combined with those of estrogen receptor α (ERα) and its ligand, estrogen. A histological study demonstrated that some cells in the AVPV co-expressed Dbp with ERα in adult female rats. Expression of ERα was not rhythmic in the AVPV, however, mRNA of Dbp in the AVPV accumulated with a robust diurnal rhythm in proestrus, but not on the first day of diestrus. Thus, these results suggest that Dbp and estrogen regulate the expression of Kiss1 in the AVPV, thereby mediating the GnRH/LH surge.

Stress Response of Prolactin-Releasing Peptide Knockout Mice as to Glucocorticoid Secretion

Prolactin‐releasing peptide (PrRP) is known to have functions in prolactin secretion, stress responses, cardiovascular regulation and food intake suppression. In addition, PrRP‐knockout (KO) male mice show obesity from the age of 22 weeks and increase their food intake. The plasma concentrations of insulin, leptin, cholesterol and triglyceride are also increased in obese PrRP‐KO mice. Fatty liver, hypertrophied white adipose tissue, decreased uncoupling protein 1 mRNA expression in brown adipose tissue and glucose intolerance were observed in obese PrRP‐KO mice. As we reported previously, PrRP stimulates corticotrophin‐releasing factor and regulates the hypothalamic‐pituitary‐adrenal axis. Therefore, it is speculated that PrRP regulates both food intake and metabolism as a stress responses. In the present study, we compared blood glucose and plasma glucocorticoid concentrations in PrRP‐KO mice, and found that PrRP‐KO mice showed higher concentrations of blood glucose and corticosterone compared to wild‐type mice after restraint stress. By contrast, there were no difference in c‐Fos expression in the paraventricular hypothalamic nucleus and plasma adrenocorticotrophic hormone concentrations between the two groups. These results suggest that the different stress responses as to glucocorticoid secretion may be induced by different responses of the adrenal glands between wild‐type and PrRP‐KO mice. Thus, we conclude that PrRP‐KO mice become obese as a result of increased food intake, a change in metabolism, and abnormal stress responses as to glucose concentration and glucocorticoid secretion.

Estrogen suppresses the stress response of prolactin-releasing peptide-producing cells.

Prolactin-releasing peptide (PrRP) is known to be produced in A1/A2 noradrenergic neurons and to mediate the stress response. Our preliminary experiment showed that PrRP neurons in the A2 region differed between males and females in terms of c-Fos expression. In addition it has been reported that estrogen receptor alpha is detectable in A2 PrRP neurons. Therefore, we speculated that the stress response of PrRP neurons is modified by estrogen. We, therefore, examined c-Fos expression in A2 PrRP neurons during the estrous cycle and found that c-Fos accumulation in PrRP neurons was significantly decreased in estrus compared with in proestrus, metestrus and diestrus. This suggests that estrogen suppresses the activation of PrRP neurons. We thus administered diethylstilbestrol (DES) to ovariectomized rats and then added restraint stress. The data clearly showed that PrRP cells in DES-administered rats significantly suppressed c-Fos accumulation induced by stress.

Cgr11 encodes a secretory protein involved in cell adhesion.

We performed comparative proteomic analyses of pituitary tumor-derived cell lines, and found a new protein, preliminarily called hydrophobestin, which was produced only in somatotrophic cells, MtT/S, but not in non-hormone-producing cells, MtT/E. Hydrophobestin is encoded by the cell growth regulatory gene, Cgr11, which is known to have growth-suppressive potential in several cell lines. We have now sought to investigate the underlying events responsible for cell growth inhibition by hydrophobestin. Immunocytochemisty revealed that hydrophobestin is localized in the Golgi apparatus of MtT/S cells and Cgr11-transfected MtT/E cells. The apparent molecular mass of the protein was determined by Westerm blot analysis of conditioned culture medium of MtT/S cells. Our data show that hydrophobestin is a secretory protein localized in the pituitary gland, adrenal gland, digestive tract, reproductive organs, and kidney. We also found that hydrophobestin promotes compact monolayer cell aggregates in PC12 cells transfected with Cgr11, however, non-transfected, vector- or EF-hand motif-deleted (DeltaEF) Cgr11-transfected PC12 cells cannot form compact cell colonies. An antibody recognizing EF-hand motifs showed strong staining in the intercellular space of both Cgr11-transfected PC12 cells and MtT/S cells (Cgr11-expressing cells). Our data suggest that hydrophobestin-mediated cell adhesion may regulate cell growth through compact cell attachment.

Blockade of PrRP attenuates MPTP-induced toxicity in mice

Prolactin-releasing peptide (PrRP) was isolated as an endogenous ligand of the orphan G-protein coupled receptor hGR3. PrRP has been shown to be involved in the regulation of food intake, stress responses, prolactin secretion and release, blood pressure, and the opioid system. Here we report that PrRP and its receptor, GPR10, were found in the mouse substantia nigra pars compacta (SNpc), the main location of dopaminergic (DA) neurons of the nigrostriatal system. We generated PrRP knockout (KO) mice, and then treated PrRP KO mice and their wild type (WT) littermates with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neuron toxin that selectively damages DA neurons in the SNpc. We found that PrRP KO mice were resistant to MPTP-induced lesions of the nigrostriatal system. These effects were further confirmed by the intracerebroventricular injection of P2L-1C, a monoclonal antibody against PrRP into mice. Taken together, our data established a critical role of PrRP in MPTP intoxication in mice.

Prolactin-Releasing Peptide

Prolactin-releasing peptide (PrRP) is found as an endogenous ligand of G-protein-coupled receptor (hGR3/UHR-1/GPR10) which is highly expressed in the anterior pituitary gland. PrRP and PrRP receptor mRNA are expressed in the brain and some peripheral tissues. In addition, PrRP positive neurons are localized in A1 and A2 noradrenalin cells in the medulla oblongata and in the dorsomedial nucleus of the hypothalamus. In addition to prolactin release, PrRP has various functions in the central nervous system, such as regulating the hypothalamic–pituitary–adrenal axis and mediating stress responses, food intake regulation, blood pressure, and the opioid system.