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Long-term diabetic patients exhibit major clinical gastrointestinal problems, such as diarrhea and constipation. In recent years, water channel protein, aquaporin 1 (AQP1) has been identified in the enteric nervous system (ENS). We have examined the pathological changes in AQP1 immunoreactive (IR) neurons in streptozotocin-induced (STZ) diabetic rats. Eight-week-old Wistar rats were injected with streptozotocin, and artificial diabetes was induced. Sixteen-week-old STZ rats were then examined with double immunofluorescence staining and ABC immunohistochemical staining. AQP1-IR neurons in STZ rats were significantly increased compared with control rats (p

Most neurons in the central nervous system and peripheral nervous system do not express water transporting protein, aquaporin (AQP). In the present study, we have demonstrated the presence of AQP1 immunoreactivity in a particular neuronal subtype in the enteric nervous system (ENS) of the rat ileum. AQP1-immunoreactive (IR) neurons simultaneously expressed a neuronal marker HuC/D. Moderate numbers of AQP1-IR neuronal somata were found in the myenteric plexus, and a very few were found in the submucosal plexus. AQP1-IR neurons can be classified as Dogiel type I cells, which have several short processes and a single long process. Many AQP1-IR fibers were found both in the myenteric and submucosal plexi. Many AQP1-IR varicose fibers were closely associated with neuronal somata in the ganglia, whereas other AQP1-IR fibers penetrated into the muscle layers. These results suggest that AQP1-IR neurons probably play a significant role within the ENS to control gut functions.

Background/Aims Nucleic acid damage by reactive nitrogen and oxygen species may contribute to inflammation-related carcinogenesis. To investigate the extent of nucleic acid damage in hepatitis C virus infection and its change after interferon treatment, we measured 8-nitroguanine and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in the liver of patients with chronic hepatitis C (CHC) before and after interferon therapy. Methods Hepatic accumulation of 8-nitroguanine and 8-OHdG was immunohistochemically evaluated in 20 CHC patients and 7 control patients with non-alcoholic fatty liver. Results Immunoreactivities of 8-nitroguanine and 8-OHdG were strongly detected in the liver from patients with CHC, but not in control livers. 8-Nitroguanine accumulation was found not only in infiltrating inflammatory cells, but also hepatocytes particularly in the periportal area. The accumulation of 8-nitroguanine and 8-OHdG increased with inflammatory grade (8-nitroguanine; P =0.0019, 8-OHdG; P =0.0009). In the sustained virological responder group after interferon therapy, 8-nitroguanine and 8-OHdG accumulation were markedly decreased in the liver (8-nitroguanine; P =0.018, 8-OHdG; P =0.018). Conclusions In this study, we demonstrated for the first time that 8-nitroguanine accumulated in the liver of patients with CHC. 8-Nitroguanine is a useful biomarker to evaluate the severity of HCV-induced chronic inflammation in relation to hepatocellular carcinoma.

Our previous study showed that the pertussis toxin-sensitive G protein, Gi2, is selectively localized in the ventricular zone of embryonic brains, where the neuroepithelial cells undergo active proliferation. In order to clarify the role of Gi2 in this site, we first administered pertussis toxin by an exo-utero manipulation method into the lateral ventricle of mouse brain at embryonic day 14.5. Examination at embryonic day 18.5 revealed that pertussis toxin-injected embryos had brains with thinner cerebral cortices, made up of fewer constituent cells. Bromodeoxyuridine labeling revealed fewer numbers of bromodeoxyuridine-positive cells in the cerebral cortices of pertussis toxin-injected embryos, suggesting impaired proliferation of neuroepithelial cells. Next we cultured neural progenitor cells from rat embryonic brains and evaluated the mitogenic effects of agonists for several Gi-coupled receptors that are known to be expressed in the ventricular zone. Among agonists tested, endothelin most effectively stimulated the incorporation of [3H]thymidine in the presence of fibronectin, via the endothelin-B receptor. This was associated with phosphorylation of extracellular signal-regulated kinase, and pertussis toxin partially inhibited both endothelin-stimulated DNA synthesis and phosphorylation of extracellular signal-regulated kinase. Injection of endothelin-3 into the ventricle of embryonic brains increased numbers of bromodeoxyuridine-positive cells in the cerebral cortex, whereas injection of an endothelin-B receptor antagonist decreased them. These findings indicate that Gi2 mediates signaling from receptors such as the endothelin-B receptor to maintain mitogenic activity in the neural progenitor cells of developing brain.

Inflammation mediated by infection is an important factor causing carcinogenesis. Opisthorchis viverrini (OV) infection is a risk factor of cholangiocarcinoma (CHCA), probably through chronic inflammation. Formation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), and expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) were assessed in the liver of hamsters infected with OV. We newly produced specific anti-8-nitroguanine antibody without cross-reaction. Double immunofluorescence staining revealed that 8-oxodG and 8-nitroguanine were formed mainly in the same inflammatory cells and epithelium of bile ducts from day 7 and showed the strongest immunoreactivity on days 21 and 30, respectively. It is noteworthy that 8-oxodG and 8-nitroguanine still remained in epithelium of bile ducts on day 180, although amount of alanine aminotransferase activity returned to normal level. A time course of 8-nitroguanine was associated with iNOS expression. Furthermore, this study demonstrated that HO-1 expression and subsequent iron accumulation may be involved in enhancement of oxidative DNA damage in epithelium of small bile ducts. In conclusion, nitrative and oxidative DNA damage via iNOS expression in hamsters infected with OV may participate in CHCA carcinogenesis.

Helicobacter pylori infection causes chronic inflammation, which can lead to gastric carcinoma. A double immunofluorescence labeling study demonstrated that the level of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) apparent in gastric gland epithelium was significantly higher in gastritis patients with H. pylori infection than in those without infection. A significant accumulation of proliferating cell nuclear antigen, a prognostic factor for gastric cancer, was observed in gastric gland epithelial cells in patients with H. pylori infection as compared to those without infection, and its accumulation was closely correlated with the formation of 8-nitroguanine and 8-oxodG. These results suggest that nitrosative and oxidative DNA damage in gastric epithelial cells and their proliferation by H. pylori infection may lead to gastric carcinoma. 8-Nitroguanine could be not only a promising biomarker for inflammation but also a useful indicator of the risk of gastric cancer development in response to chronic H. pylori infection.

Components of the multidrug resistance-associated protein (mrp) family mediate the adenosine triphosphate (ATP)-dependent transport of conjugated organic anions in the liver. Of these, mrp1 and mrp2 have been shown to have similar substrate specificity and nucleotide sequence. The intracellular localization and distribution of mrp1 under normal condition and cholestasis have not been as yet completely elucidated. To clarify this point, in the present study we evaluated the intracellular localization of mrp1 in rat liver and kidney after bile duct ligation (BDL). Bile duct was ligated in Wistar rats. Sequential staining of mrp1 by immunofluorescence was carried out in rat liver and kidneys 1, 3, and 5 days after bile duct ligation using confocal laser scanning microscopy. Weak granular staining of mrp1 was observed in cytoplasm of control rat hepatocytes. In addition to increased cytoplasm staining of mrp1, belt-and granule-like staining of mrp1 in basolateral membrane of hepatocytes was also shown after BDL. Furthermore, mrp1 immunofluorescence increased over time after BDL. No specific immunoflurescence of mrp1 was detected in control rat kidney. However, mrp1-positive staining was observed in epithelia of some renal tubules after BDL. This study showed that mrp1 immunofluorescence increased in hepatocyte basolateral membrane and cytoplasm and epithelia of some renal tubules after BDL. This increased mrp1 expression may be an adaptive response to impairment of hepato-biliary organic anion transport during obstructive cholestasis.

Most of the nerve growth factor (NGF) protein in the rat and mouse brain is readily extractable in the presence of guanidine hydrochloride as is the case of brain-derived neurotrophic factor. In the present study, we measured amounts of NGF that could be extracted in the presence and absence of 1 M guanidine hydrochloride from various regions of the brains of male and female mice. About 14% of the total NGF in the hippocampus from female mice at 4 months of age could be extracted without 1 M guanidine hydrochloride (designated loosely bound NGF; about 32% in the rat hippocampus) and the remainder only in its presence (designated tightly bound NGF). The molecular masses of the NGF-immunoreactive protein in both cases were ∼ 14 kDa. There were significant differences in respective concentrations of total NGF (the loosely bound plus tightly bound NGF) in the hypothalamus and hypophysis, but not in other brain regions, between male and female mice at 4 months of age. However, levels of loosely bound NGF in the cerebellum and olfactory bulb from males were significantly higher than those in the same regions from females. This difference resulted in two-fold higher ratios of the concentrations of loosely bound to total NGF in males as compared to females. On the other hand, the ratio in the hypophysis was close to unity in both sexes. The concentrations of loosely bound NGF in the hippocampus and cerebral cortex decreased slightly with age in both males and females. Levels of loosely bound NGF increased significantly from 2 to 12 months after birth in the whole brain, olfactory bulb, cerebellum, hypothalamus and hypophysis to a greater extent in males than in females. Thus, it is suggested that high ratios of loosely bound to total NGF in selected regions of brains from male mice are due to an enhanced conversion from tightly to loosely bound form, which is considered to be regulated by androgens (see Brain Res. 322, 112–117, Brain Res. 322, 1990 ). They may also influence the total NGF expression.

Age-related changes in the levels of brain-derived neurotrophic factor (BDNF) in selected regions of brains from rats, normal mice and senescence-accelerated mice were compared to those of nerve growth factor (NGF) and neurotrophin-3 (NT-3). The concentration of BDNF increased with age in the rat hippocampus while it decreased in the rat cerebral cortex. The level of BDNF in the hippocampus from aged rats was about 260%, of that in the same region from young adult rats. A strong staining with antibodies specific for BDNF was observed in the hilus of the dentate gyrus in the hippocampus from aged rats. By contrast, BDNF levels were significantly lower in four brain regions from aged rats as compared to young adult rats (30, 56, 52 and 52%, lower in the septum, cerebral cortex, cerebellum and striatum, respectively). Patterns of age-related changes in the level of BDNF in the mouse hippocampus. cerebral cortex, cerebellum and olfactory bulb were similar to those in the respective regions from rats. In rats, the concentration of NGF decreased with age in the cerebral cortex but remained unchanged in the hippocampus, cerebellum and olfactory bulb. In mice, levels of NGF increased in all four brain regions from 1 to 18 months after birth. The concentrations of NT-3 increased and decreased with age in the rat cerebral cortex and cerebellum, respectively, while minimal changes were observed in the rat hippocampus and olfactory bulb as was also true in mice. In senescence-accelerated mice with memory disturbances, no marked increases in levels of NGF and BDNF in the hippocampus and in the level of NT-3 in the cerebral cortex were found. Thus, increases in levels of BDNF and NT-3 occurred in the murine hippocampus and cerebral cortex, respectively, during normal aging, but not during aging of mice with pathological changes.

Carbon monoxide (CO) is a gas that can permeate biological membranes and it has been suggested that the gas plays a signaling role in the brain by activating guanylyl cyclase (sGC). CO is generated by heme oxygenase during the conversion of heme to biliveridn. In this study, we raised an antiserum against the chemically synthesized amino-terminal fragment of heme oxygenase-2 (HO-2) and studied the distribution of this enzyme in the rat cerebellum by an immunocytochemical method. Immunoreactivity specific for HO-2 was observed only in neurons. In the Purkinje cells and the basket cells of the cerebellum, immunoreactivity was detected in the dendrites and the somata but not in the axon terminalis, suggesting that CO might be liberated primarily from the dendrites and somata rather than from the axons in this region of the rat brain.

Gamma-aminobutyric acid (GABA) and glycine are known as major inhibitory neurotransmitters in the intermediolateral nucleus of the spinal cord. Distribution and density of GABA immunoreactive axon varicosities and glycine receptor immunoreactive dendrites and somata in the intermediolateral nucleus were examined by immuno-electronmicroscopy. GABA immunoreaction was observed in the axon varicosities of axo-dendritic and axo-somatic synapses. Glycine receptor immunoreaction was seen in association with the postsynaptic membrane of dendrites and soma. GABA immunoreactive axon varicosities were larger (1.01 +/- 0.49 x 1.20 +/- 0.38 microns) than axon varicosities presynaptic to glycine receptors (0.72 +/- 0.22 x 0.98 +/- 0.33 microns). The density of GABA immunoreactive axon varicosities was 3.65/100 microns 2 and that of glycine receptor immunoreactive synapses was 4.78/100 microns 2. A subpopulation of GABA immunoreactive axons (42%) made synaptic contact with glycine receptor immunoreactive dendrites or soma, indicating the coexistence of GABA and glycine.

Changes in parvalbumin-like immunoreactivity were studied during ontogenetic development of the central auditory system of the Mongolian gerbil ( Meriones unguiculatus ). The nucleus of the trapezoid body contained cells that could be stained on the day of birth while in the superior olivary complex, prominent staining of cell somata was only found at P15 (postnatal day 15). Neurons located in the ventral nucleus, in the dorsal nucleus of the lateral lemniscus and in the inferior colliculus developed parvalbumin immunoreactivity mostly between P11–P15 (ventral nucleus), P15–P19 (dorsal nucleus) and P15–P19 (inferior colliculus), respectively. The accumulation of PV correlates reasonably well with the functional maturation of the neurons in these areas.

The coexistence of glycine- and PV-immunoreactivities was studied immunocytochemically in the nuclei of the superior olive, trapezoid body, cochlea and lateral lemniscus. All of the PV-immunoreactive neurons in the nuclei of the superior olive and trapezoid body were immunoreactive to glycine but not to GABA. In the dorsal cochlear nucleus, PV-positive neurons were sometimes immunoreactive to glycine. In the ventral nucleus of the lateral lemniscus, PV-positive cells were immunoreactive neither to glycine nor to GABA. Consequently, it was concluded that PV-immunoreactivity was distributed not only in the GABAergic neurons, but also in the glycinergic neurons and possibly in wider neuronal populations.

In our previous report, we have shown the sex difference in the concentration of the beta-subunit of nerve growth factor (beta-NGF) in the neural and paraneural tissues of mice. In this investigation, we examined the effects of castration of adult males, and of neonatal and/or adult treatments with testosterone on levels of beta-NGF in the several tissues of mice. Castration caused a marked reduction in the levels of serum testosterone and of beta-NGF in the brain, spinal cord and submandibular glands, but not in the pancreas and kidneys. Continuous infusion of testosterone for one week into adult males that had been castrated at 2 months of age restored the level of beta-NGF in the three tissues mentioned above. A single injection of testosterone to 5-day-old female pups to masculinize the brain gave no effect on the level of beta-NGF in any tissue dissected after 4 months. A one-week infusion of testosterone into adult females slightly increased levels of beta-NGF in the brain and spinal cord, but the same treatment of adult females given in advance a single dose of testosterone at 5 days of age caused a significant increase in its levels over those of untreated females. These results suggest that neonatal and adult exposures to testosterone can influence the endogenous concentration of beta-NGF in the brain and spinal cord.

The alpha-subunit of a GTP-binding protein Go (Go alpha), is a new marker protein of neurons and neuroendocrine cells. In our previous report, this protein was shown to be also distributed in the cardiac muscle of young rats, although the cardiac muscle is traditionally thought to be a non-neuronal tissue. To evaluate this unusual finding, expression of other neural marker proteins in the heart was examined. Immunohistochemical studies using antibodies against gamma-enolase, neurofilament and Go alpha protein revealed localization of these neural markers only in neurons and neuroendocrine cells in adult rats. In young rats, however, these neural markers were localized not only in these cells but also in the cardiac muscle. An enzyme immunoassay study of enolase isozymes showed that the heart of young rats was rich in neuron-specific gamma-enolase rather than in muscle-specific beta-enolase, while the heart of adult rats was deprived of the former and rich in the latter. These results suggest that the heart of young rats should be understood as a neuroendocrine tissue as well as a muscular tissue.

Cerebellar cortical neurons immunoreactive to anti-γ-aminobutyric acid (GABA) antibody were examined in the rat. In addition to the Purkinje, Golgi, basket and stellate cells, spindle-shaped cells lying just below the Purkinje cell layer were found to be strongly immunoreactive to the antibody. By the combination of immunofluorescence and hematoxylin stainings, these GABA-positive cells were shown to be the Lugaro cells. Unlike the immunopositive small Golgi cell, the pale cell was not immunoreactive to the antibody.

gamma-Enolase has been believed to be distributed only in the neurons and it was frequently labelled as neuron-specific enolase. However, recent precise studies have suggested a wider distribution of the protein. It can also be found in neuroendocrine cells, some mesodermal tissues, and some malignant tumors originating from tissues without the antigen in a normal condition. In early rat embryos, before the formation of neural tissues, a sensitive immunoassay system revealed a substantial amount of gamma-enolase, though it is not yet clear where the antigen is located. In the present study, tissue distribution of gamma-enolase in early human embryos was studied using an immunohistochemical method and it was suggested that the protein is present not only in neural tissue primordium but also in most tissues in the youngest embryo of 6.3 mm crown-rump length. Many of the immunoreactive non-neural tissues, however, lost immunoreactivity with the advancement of the embryonic stage while neural tissues became more intensely stained. In the embryo of 24 mm length, the staining pattern was almost the same as that of reported adult men. In embryonic tissues such as notochord and mesonephros, which disappear in the due course of growth, the antigen was also found. These findings will suggest that the antigen is rather common in undifferentiated tissues but then localizes in neural elements with advancing age. Our findings may be useful in explaining why early rat embryos, before the formation of neural tissues, showed a considerable amount of gamma-enolase and why many undifferentiated tumors, originating from tissues which did not have the antigen in normal condition, revealed the antigen.

The tissue distribution of muscle-type alpha beta and beta beta enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the alpha and to the beta subunit of enolase, and beta-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both alpha beta and beta beta enolases. The beta beta enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The alpha beta enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The beta beta enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the alpha alpha and alpha beta enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of beta beta enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum longus (about 800 pmol/mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of alpha alpha (2 pmol/mg) and alpha beta (80 pmol/mg) usually showed a slight increase by the treatment (alpha alpha, 7 pmol/mg; alpha beta, 100 pmol/mg after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of beta beta enolase is closely correlated with the functional state of the muscle tissue.

The distribution of 3 forms of rat enolase (alpha alpha, alpha gamma and gamma gamma forms), including nervous system-specific forms (alpha gamma and gamma gamma), was determined in various tissues with a sensitive enzyme immunoassay system. The brain and spinal cord contained more than 100 pmol/mg protein of the alpha alpha, alpha gamma and gamma gamma enolases. Organs such as the lungs, heart, spleen, liver, and kidney contained similarly high levels of alpha alpha enolase, but these tissues contained alpha gamma and gamma gamma enolases at levels less than 1% of the central nervous tissues. High levels of the alpha gamma (greater than 10 pmol/mg) and the gamma gamma (greater than 1.5 pmol/mg) forms were found in rectum, bladder, and uterus. In gut, major portions of the nervous system-specific forms were localized in the muscle layers. Skeletal muscle and diaphragm, which are composed of striated muscle, contained low levels of 3 forms of enolase. Megakaryocytes separated from the suspension of bone marrow contained 11.3, and 0.53 amol/cell of the alpha alpha and gamma gamma enolases, respectively, with little, if any, of the alpha gamma form.