Effect of Aralia Elata on the Expression of Hyperosmolarity-induced TonEBP Protein and Inflammatory Mediators in Corneal Epithelial Cells

Purpose@#To investigate the effect of Aralia elata (AE) on hyperosmolar stress-induced tonicity response enhancer-binding protein (TonEBP) expression and changes in the levels of proinflammatory cytokines in immortalized human corneal epithelial cells (hCECs). @*Methods@#Immortalized hCECs were cultured with either 5 or 10 μg/mL AE for 24 hours, and the medium then changed to a hyperosmotic medium (500 mOsM/L). After hyperosmolar treatment, cell viability and wound-healing assays were performed, and cell proteins subjected to Western blot analysis, immunocytochemistry for TonEBP and NF-κB, and tests measuring changes in the levels of oxidative stress markers and inflammatory mediators. @*Results@#AE pretreatment ameliorated hyperosmolarity-induced cell death and the delay in wound-healing in a dose-dependent manner. AE inhibited TonEBP and phospho-NF-κB p65 subunit upregulation. AE significantly decreased the expression levels of Bax, 4-HNE, and IL-1β; but increased those of Bcl-2, Bcl-xl, and Gpx. @*Conclusions@#AE increased cell viability and wound-healing, and inhibited the hyperosmolar stress-induced upregulation of TonEBP and NF-κB. AE may be useful for treatment of patients with certain ocular surface diseases.

Glutamine Supplementation Ameliorates Chronic Stress-induced Reductions in Glutamate and Glutamine Transporters in the Mouse Prefrontal Cortex

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS.

Quercetin induces cell death in cervical cancer by reducing O-GlcNAcylation of adenosine monophosphate-activated protein kinase / 대한해부학회지

Hyper-O-GlcNAcylation is a general feature of cancer which contributes to various cancer phenotypes, including cell proliferation and cell growth. Quercetin, a naturally occurring dietary flavonoid, has been reported to reduce the proliferation and growth of cancer. Several reports of the anticancer effect of quercetin have been published, but there is no study regarding its effect on O-GlcNAcylation. The aim of this study was to investigate the anticancer effect of quercetin on HeLa cells and compare this with its effect on HaCaT cells. Cell viability and cell death were determined by MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assays. O-GlcNAcylation of AMP-activated protein kinase (AMPK) was examined by succinylated wheat germ agglutinin pulldown and immunoprecipitation. Immunofluorescence staining was used to detect the immunoreactivitiy of O-linked N-acetylglucosamine transferase (OGT) and sterol regulatory element binding protein 1 (SREBP-1). Quercetin decreased cell proliferation and induced cell death, but its effect on HaCaT cells was lower than that on HeLa cells. O-GlcNAcylation level was higher in HeLa cells than in HaCaT cells. Quercetin decreased the expression of global O-GlcNAcylation and increased AMPK activation by reducing the O-GlcNAcylation of AMPK. AMPK activation due to reduced O-GlcNAcylation of AMPK was confirmed by treatment with 6-diazo-5-oxo-L-norleucine. Our results also demonstrated that quercetin regulated SREBP-1 and its transcriptional targets. Furthermore, immunofluorescence staining showed that quercetin treatment decreased the immunoreactivities of OGT and SREBP-1 in HeLa cells. Our findings demonstrate that quercetin exhibited its anticancer effect by decreasing the O-GlcNAcylation of AMPK. Further studies are needed to explore how quercetin regulates O-GlcNAcylation in cancer.

Myeloid-specific SIRT1 Deletion Aggravates Hepatic Inflammation and Steatosis in High-fat Diet-fed Mice

Sirtuin 1 (SIRT1) is a mammalian NAD+-dependent protein deacetylase that regulates cellular metabolism and inflammatory response. The organ-specific deletion of SIRT1 induces local inflammation and insulin resistance in dietary and genetic obesity. Macrophage-mediated inflammation contributes to insulin resistance and metabolic syndrome, however, the macrophage-specific SIRT1 function in the context of obesity is largely unknown. C57/BL6 wild type (WT) or myeloid-specific SIRT1 knockout (KO) mice were fed a high-fat diet (HFD) or normal diet (ND) for 12 weeks. Metabolic parameters and markers of hepatic steatosis and inflammation in liver were compared in WT and KO mice. SIRT1 deletion enhanced HFD-induced changes on body and liver weight gain, and increased glucose and insulin resistance. In liver, SIRT1 deletion increased the acetylation, and enhanced HFD-induced nuclear translocation of nuclear factor kappa B (NF-kappaB), hepatic inflammation and macrophage infiltration. HFD-fed KO mice showed severe hepatic steatosis by activating lipogenic pathway through sterol regulatory element-binding protein 1 (SREBP-1), and hepatic fibrogenesis, as indicated by induction of connective tissue growth factor (CTGF), alpha-smooth muscle actin (alpha-SMA), and collagen secretion. Myeloid-specific deletion of SIRT1 stimulates obesity-induced inflammation and increases the risk of hepatic fibrosis. Targeted induction of macrophage SIRT1 may be a good therapy for alleviating inflammation-associated metabolic syndrome.

FK506 reduces calpain-regulated calcineurin activity in both the cytoplasm and the nucleus / 대한해부학회지

Excessive immune responses induced by ischemia-reperfusion injury (IRI) are known to lead to necrotic and apoptotic cell death, and calcineurin plays a major role in this process. Calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT), permitting its translocation into the nucleus. As a result, calcineurin promotes the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha. The overproduction of pro-inflammatory cytokines causes renal cell death. Calcineurin activity is regulated by calpain, a cysteine protease present in the nucleus. Calpain-mediated proteolysis increases the phosphatase activity of calcineurin, resulting in NFAT dephosphorylation. This process has been studied in cardiomyocytes but its role in renal IRI is unknown. Thus, we examined whether calpain regulates calcineurin in renal tubule nuclei. We established an in vivo renal IRI model in mice and identified the protective role of a calcineurin inhibitor, FK506, in this process. Calcineurin is expressed in the nucleus, where it is present in its calpain-cleaved form. FK506 reduced nuclear expression of calcineurin and prevented calcineurin-mediated NFAT activation. Our study shows clearly that FK506 reduces calpain-mediated calcineurin activity. Consequently, calcineurin could not maintain NFAT activation. FK506 reduced renal cell death by suppressing the transcription of pro-inflammatory cytokine genes. This study provides evidence that FK506 protects against inflammation in a renal IRI mouse model. We also provided a mechanism of calcineurin action in the nucleus. Therefore, FK506 could improve renal function by decreasing calcineurin activity in both the cytoplasm and the nucleus of renal tubule cells.

FK506 reduces calpain-regulated calcineurin activity in both the cytoplasm and the nucleus / 대한해부학회지

Excessive immune responses induced by ischemia-reperfusion injury (IRI) are known to lead to necrotic and apoptotic cell death, and calcineurin plays a major role in this process. Calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT), permitting its translocation into the nucleus. As a result, calcineurin promotes the release of pro-inflammatory cytokines, such as tumor necrosis factor-alpha. The overproduction of pro-inflammatory cytokines causes renal cell death. Calcineurin activity is regulated by calpain, a cysteine protease present in the nucleus. Calpain-mediated proteolysis increases the phosphatase activity of calcineurin, resulting in NFAT dephosphorylation. This process has been studied in cardiomyocytes but its role in renal IRI is unknown. Thus, we examined whether calpain regulates calcineurin in renal tubule nuclei. We established an in vivo renal IRI model in mice and identified the protective role of a calcineurin inhibitor, FK506, in this process. Calcineurin is expressed in the nucleus, where it is present in its calpain-cleaved form. FK506 reduced nuclear expression of calcineurin and prevented calcineurin-mediated NFAT activation. Our study shows clearly that FK506 reduces calpain-mediated calcineurin activity. Consequently, calcineurin could not maintain NFAT activation. FK506 reduced renal cell death by suppressing the transcription of pro-inflammatory cytokine genes. This study provides evidence that FK506 protects against inflammation in a renal IRI mouse model. We also provided a mechanism of calcineurin action in the nucleus. Therefore, FK506 could improve renal function by decreasing calcineurin activity in both the cytoplasm and the nucleus of renal tubule cells.

Triamcinolone Acetonide Prevents Enhancement of Hypoxia-induced Neuronal and Inducible Nitric Oxide Synthases in the Retinas of Rats with Oxygen-induced Retinopathy

PURPOSE: We investigated whether oxygen-induced retinopathy (OIR) results in changes in the protein expression of neuronal and inducible nitric oxide synthases (nNOS and iNOS, respectively) in rat model of OIR. In addition, we evaluated whether treatment of rats with triamcinolone acetonide (TA) prevents this response. METHODS: To promote OIR, Sprague-Dawley rats were exposed to hyperoxia from postnatal day 2 (P2) to P14. They were then returned to normoxia after P15. TA was injected into the right vitreous of P15 rats, while saline was injected into the left vitreous. At P18 the expression of nNOS and iNOS was determined using Western blotting and immunostaining techniques in retinas obtained from control rats. RESULTS: In P18 OIR rats, the abundance of nNOS and iNOS protein was significantly increased compared with controls. These increases were not observed in the retinas of rats treated with TA. The change in expression of nNOS and iNOS were specific to parvalbumin and glial fibrillary acidic protein-positive cells. Treatment with TA prevented the increased expression of nNOS and iNOS in all samples. CONCLUSIONS: Hypoxia upregulates expression of nNOS and iNOS in OIR rat retinas, which is can be prevented by treatment with TA.

Proapoptotic role of nuclear clusterin in brain / 대한해부학회지

Clusterin (CLU) is a multifunctional glycoprotein that has secretory and nuclear isoforms. The two isoforms are known to play opposite roles in cell survival/death. In this review, we summarize recent progress on the pro-apoptotic function of nuclear CLU in vitro and in vivo and discuss previous reports on the role of CLU in brain damage and neurodegeneration.

Resveratrol activates AMPK and suppresses LPS-induced NF-kappaB-dependent COX-2 activation in RAW 264.7 macrophage cells / 대한해부학회지

AMP-activated protein kinase (AMPK), an enzyme involved in energy homeostasis, regulates inflammatory responses, but its precise mechanisms are not fully understood. Recent evidence has shown that resveratrol (RES), an AMPK activator, reduces prostaglandin E2 production in lipopolysaccharide (LPS)-treated microglia. Here, we examined the effect of RES on nuclear factor kappa B (NF-kappaB) dependent cyclooxygenase (COX)-2 activation in LPS-treated RWA 264.7 macrophages. We found that treatment with RES increased AMPK activation. AMPK and acetyl CoA carboxylase phosphorylation were attenuated in cells treated with LPS+RES, compared to cells treated with LPS alone. RES inhibited tumor necrosis factor (TNF)-alpha and TNF receptor 1 in LPS-treated cells. Finally, RES inhibited LPS-induced NF-kappaB translocation into the nucleus and COX-2 expression. Moreover, the effects of 5-aminoimidazole-4-carboxamide ribose and compound C were consistent with the effects of RES in LPS-treated cells. Taken together, these results suggest that the anti-inflammatory action of RES in RAW 264.7 macrophages is dependent on AMPK activation and is associated with inhibition of the LPS-stimulated NF-kappaB-dependent COX-2 signaling pathway.

Phosphorylation of 14-3-3zeta at serine 58 and neurodegeneration following kainic acid-induced excitotoxicity / 대한해부학회지

Oxidative stress-induced cell death leads to phosphorylation of 14-3-3zeta at serine 58. 14-3-3zeta is detected at significant levels in cerebrospinal fluid after kainic acid (KA)-induced seizures. Here we examined temporal changes in 14-3-3zeta phosphorylation in the hippocampus and amygdala of mice after KA treatment. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. We observed an increase in TUNEL and Fluoro-Jade B (FJB)-stained neurons in the hippocampus and amygdala of KA-treated mice. Phospho (p)-14-3-3zeta and p-JNK expression was increased in the hippocampus 2 and 6 h after KA treatment, respectively. In immunohistochemical analysis, p-14-3-3zeta-positive cells were present in the CA3 region of the hippocampus and the central nucleus of amygdala (CeA) of KA-treated mice. Thus, phosphorylation of 14-3-3zeta at serine 58 may play an important role in KA-induced hippocampal and amygdaloid neuronal damage.

Ethanol down regulates the expression of myelin proteolipid protein in the rat hippocampus / 대한해부학회지

It is well known that chronic ethanol treatment affects the synthesis of RNA and protein in the brain and the maintenance and function of nervous system. The changes in myelination-related genes are most prominent in human alcoholics. Previously, our cDNA microarray study showed altered Proteolipid protein (PLP), a major protein of central myelin. The present study aimed to gain more understanding of the expression of PLP after chronic ethanol treatment. Male Sprague-Dawley rats were daily treated with ethanol (15% in saline, 3 g/kg, i.p.) or saline for 14 days. Messenger RNAs from hippocampus of each group were subjected to cDNA expression array hybridization to determine the differential gene expressions. Among many ethanol responsive genes, PLP was negatively regulated by ethanol treatment, which is one of the most abundant proteins in the CNS and has an important role in the stabilization of myelin sheath. Using northern blot and immunohistochemical analysis, we showed the change in expression level of PLP mRNA and protein after ethanol treatment. PLP mRNA and protein were decreased in hippocampus of rat with chronic ethanol exposure, suggesting that ethanol may affect the stabilization of myelin sheath through the modulation of PLP expression and induce the pathophysiology of alcoholic brain.

Streptozotocin-induced Diabetes Increases the Adiponectin-mediated AMP-activated Protein Kinase Cascade in the Hippocampus of Mice / 대한해부학회지

Adiponectin is an adipocyte-derived protein with anti-diabetic and anti-angiogenesis properties that improves both glucose metabolism and insulin resistance via the adenosine monophosphate-activated protein kinase (AMPK) cascade. Diabetic cognitive deficits are correlated with dysregulation of energy metabolism in the hippocampus. In the present study, we investigated the expression of adiponectin-mediated AMPK cascade proteins in the hippocampus of streptozotocin (STZ)-induced diabetic mice. Diabetes was induced by STZ (55 mg/kg) injection intraperitoneally. Twenty-four weeks after induction of diabetes, mice were sacrificed. Results showed that decreased serum adiponectin levels and increased expression of hippocampal adiponectin receptor 1 (AdipoR1) was expressed in diabetic mice. Phosphorylated AMPK, acetyl CoA carboxylase (ACC), and eNOS expression levels were increased in the hippocampus of diabetic mice. The immunoreactivity of glucose transporter 1 in the endothelium of hippocampal blood vessels was also increased. These results indicate that adiponectin-mediated AMPK cascade activation may play a role in catabolic process that is involved in diabetic neurodegeneration.

Cytoprotective Effects of Serum Hormone Deprivation against Glutamate Toxicity in HT22 Mouse Hippocampal Cells / 대한해부학회지

Reactive oxygen species (ROS) are important signaling molecules or mediators in many cellular responses, including the oxidative-burst defense response. Certain hormones are neuroprotective because they are modulators of neuronal activity or ROS scavengers. We have examined the effect of a hormone-free condition on ROS levels following glutamate-induced excitotoxicity in the mouse hippocampal HT22 cell line. We show that hormone starvation slightly elevates ROS and that continuous low concentrations of ROS induce expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1). In addition, N-acetyl-L-cysteine (NAC) restores the expression of ERK1/2 protein in hormone-starved HT22 cells. These findings suggest that whereas high-dose ROS are cytotoxic and lead to tissue damage in the brain low-dose ROS may act in neuroprotective signaling.

Immunohistochemical Study of Glucose Transporters 1 and 3 in the Mouse Hippocampus after Kainic Acid Treatment / 대한해부학회지

Seizure activity increases glucose utilization within the brain in response to neuronal injury. In this study, we investigated the expression of two brain glucose transporter (GLUT) proteins, GLUT1 and GLUT3, in the mouse hippocampus after kainic acid (KA) treatment. Forty-eight hours after KA (30 mg/kg) injection, mice were sacrificed and a histological evaluation of KA-treated hippocampus revealed cell death using cresyl violet staining and immunohistochemistry for caspase-3. In KA-treated hippocampus, reactive astrocytic changes were confirmed by increased immunoreactivity of glial fibrillary acidic protein (GFAP). Enhanced GLUT1-positive endothelial cells were present in the hippocampus after KA treatment. However, GLUT3-positive neurons were not localized to the KAtreated hippocampus. In particular, although GLUT-3 was not expressed in the hippocampus, pronounced GLUT3- positive cells were observed in the hypothalamic paraventricular nucleus (PVN), which controls energy metabolism. Thus, these results indicate that changes in endothelial GLUT1 and neuronal GLUT3 levels in response to neural injury may play important roles in neuroprotection against brain excitotoxicity.

Involvement of VEGF in both Cell Apoptosis and Survival in the Retina of Type 2 Diabetic OLETF Rats / 대한해부학회지

Vascular endothelial growth factor (VEGF) is closely involved in early retinal pathology of diabetes, including blood-retinal barrier breakdown, pericyte loss, neuro-retinal apoptosis, and cell proliferation. This study examines the involvement of VEGF in cell apoptosis and survival in the retina of animals with type 2 diabetes. We used retinas from 28-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of spontaneous type 2 diabetes, and Long-Evans Tokushima Otsuka (LETO) rats as controls. In parallel with evidence for pericyte loss, we found cell proliferation, apoptosis, and endothelial nitric oxide synthase (eNOS) (an indicator of endothelial cell proliferation/survival) and VEGF overexpression in the OLETF-retina, compared to control LETO. Furthermore, apoptotic signals were partly co-localized to only VEGF-positive cells in the OLETF-retina, but no apoptotic signals were found in VEGF- and eNOS-double-positive cells. These results suggest that upregulated VEGF is involved in apoptosis and eNOS-dependent cell survival in the retinas of type 2 diabetic rats.

The Changes in the Expression of gamma-Aminobutyric Acid Related Enzymes in the Mouse Hippocampus Following Ketogenic Diet / 대한해부학회지

The ketogenic diet (KD) has been used to treat intractable childhood epilepsy. However, its mechanism of action remains unknown. In the present study, we examined the effects of KD on the expression of multiple constituents of the GABAergic system in the hippocampus through immunohistochemistry and northern blot analysis. From the results, we have shown that KD increased expression of GABA and decreased GABA transporter1 (GABATp) and GABA transaminase (GABA-T) mRNA levels in the hippocampus. These results suggest that the neuroinhibitory effect of KD may be mediated, at least in part, by the increment of GABAergic activity in the hippocampus. KD may increase the GABA levels in the synaptic space by limiting GABA reuptake and in the presynaptic nerve terminal by inhibiting GABA degradation.

Alterations of Tyrosine Hydroxylase and Choline Acetyltransferase in the Retina of the Diabetic Rat / 대한해부학회지

To investigate the effect of hyperglycemia on the visual system, we investigated the retinal dopaminergic and cholinergic systems using tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the rat retinas of streptozotocin (STZ)-induced diabetes. Diabetes was induced by a single intraperitoneal injection of STZ (50 mg/kg) to Sprague-Dawley rats (250~300 g). We first analyzed morphologic thickness changes in the several retinal layers of 6-week-old control and STZ-diabetic rats after H & E staining. To confirm whether TH and ChAT protein expressions changed, we carried out immunohistochemistry analysis and Western blotting. After induction of diabetes, significant changes were not shown in the retinal thickness at 6 weeks. TH and ChAT immunoreactivities were clearly detected in amacrine cells and sublaminas in the inner retina of both control and diabetic rats, showing continuously reduced positive amacrine cells in the retinas during diabetes. In addition, the decline in TH and ChAT protein expression was already present to a significant extent in the retina at 6 weeks in early diabetes. Our present study demonstrates the possibility that the observed alterations in TH and ChAT in the diabetic retina may cause the visual system changes in the retinal pathophysiology associated with diabetes mellitus.

Two-phalanged Fifth Toe in Korean Children / 체질인류학회지

Radiographic research was performed to know the frequency of two-phalanged fifth toe and its relation to presence of the ossification centers in normal Korean children. Previous study showed more than 74% of the incidence in adulthood and less than 30% in childhood. Fifty children (33 male and 17 female, aged 2 to 15; mean age 9.6) were studied by plain foot radiographs focused on the fifth toe. In the 3~8 yr old 20 subjects, secondary ossification center of distal phalangeal bone was seen as a ossicle (small bone) placed at proximal to the distal phalanx. Secondary ossification center of middle phalangeal bone and the bony shaft of the phalanx was hard to distinguish. So keeping up the objectivity, regardless of distinguishable ossification center or the bony shaft of phalanges, ossicles seen on the 5th toe was counted to classify the presumptive type of the toe. Epiphyseal ossification center of proximal phalanx was excluded from the count. There were three types of the fifth toe which has 2 ossicles to 4 ossicles. Overall incidence of the type of 2 ossicles was 24% (12/50). Above 12 yr old group the incidence was 61% (11/18), and above 13 yr old group the incidence was 75% (9/12). The incidence of biphalangism came closer to the adult's after late childhood. This finding represent that progress of biphalangealization completed after late childhood. It seems that the progress starts earlier than 3 yr old. We made the hypothesis by the incidence of 30% (6/20) of the type which has 4 ossicles on the fifth toe at 3~8 yr old group. Four ossicles might be a secondary ossification center of distal phalanx and the bony shaft of distal, middle and proximal phalanx. They might form a distal interphalageal joint and the triphalangeal toe. To know more about the morphogenesis of biphalalngeal 5th toe, further progressive study in childhood is needed.

Differential Effects of Antipsychotic Drugs on Dopamine D1 and D2 Receptor mRNAs in the Rat Brain / 대한해부학회지

The principal aim of this study was to determine the effects of antipsychotics (haloperidol, sulpiride, and clozapine) on regulating dopamine (DA) D1 and D2 receptor mRNA levels in the rat caudate putamen (CPu), nucleus accumbens (NAc), and olfactory tubercle (OTu). Twenty male Sprague-Dawley rats (250 g) were treated with haloperidol (1mg/mL), sulpiride (40 mg/mL), clozapine (20 mg/mL), and the control group received only water. Drugs were administered orally for 4 weeks. Antipsychotic drugs had differential effects on DA D1 and D2 receptor gene expression. Haloperidol and sulpiride induced an increase of DA D1 and D2 receptor mRNA levels in the rat CPu, OTu, and NAc; haloperidol caused a greater increase than sulpiride. However, clozapine treatment had less effect on DA receptor mRNAs levels in the same area. Antipsychotic drugs differentially upregulated the expression of DA D1 and D2 receptor mRNAs in the rat brain. These changes may be related, at least in part, to changes of DA concentration following antipsychotics treatment.

Expression of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in human placenta

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC1 ) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth.