Short-term hypoxia transiently increases dopamine ß-hydroxylase immunoreactivity in glomus cells of the rat carotid body.

Under long-term hypoxia, noradrenaline (NA) content in the carotid body (CB) increases, suggesting that NA plays an important role in CB chemotransduction. However, it is unknown whether short-term hypoxia upregulates NA biosynthesis in CB. Therefore, we examined dopamine ß-hydroxylase (DBH) expression in the CB of rats exposed to hypoxia (10% O(2)) for 0 to 24 hr with immunoblotting and immunohistochemistry. Using immunoblotting, the signal intensity for DBH appeared to be the most intense in rats exposed to hypoxia for 12 hr. Using immunohistochemistry, DBH immunoreactivity was observed in the cytoplasm of some glomus cells and varicosities in controls and rats exposed to hypoxia for 6 hr. In rats exposed to hypoxia for 12 hr, DBH immunoreactive intensities in DBH-positive glomus cells were significantly higher compared with controls (p<0.05). In the CB of rats exposed to hypoxia for 18 and 24 hr, DBH immunoreactive intensities in DBH-positive glomus cells were significantly lower than that of rats exposed to hypoxia for 12 hr (p<0.05). These results demonstrate that DBH immunoreactivity is transiently increased in glomus cells by short-term hypoxia, suggesting that NA biosynthesis is transiently facilitated in glomus cells at an early stage of hypoxia.

Morphological development and expression of neurotrophin receptors in the laryngeal sensory corpuscles.

Morphological development of sensory structures in the laryngeal mucosa of postnatal rats was observed by use of immunohistochemistry for protein gene-product 9.5 (PGP9.5). Moreover, expression changes of high affinity neurotrophin receptors, TrkA, TrkB and TrkC, and low affinity neurotrophin receptor p75(NTR) were examined to elucidate the relationship to morphogenesis. Intraepithelial nerve endings and parent axons of the laminar endings with immunoreactivity for PGP9.5 have already appeared in the rat on embryonic day 18 (E18) as well as solitary chemoreceptor cells in the glottic cleft. According to neurotrophin receptors, TrkA immunoreactivity were observed on and after postnatal week 3 (3W) in the nervous sensory structures, that is, free nerve endings, laminar endings and sub- and intragemmal plexuses of the taste buds. In the laminar endings, TrkC immunoreactivity was also observed on and after 3W. According to the laryngeal sensory cells, the solitary chemoreceptor cells were immunoreactive to TrkA, TrkB, and TrkC on and after postnatal day 3 (P3). In the taste buds in arytenoid region, taste cells were immunoreactive for TrkA, TrkB, and TrkC on and after 3W, P14, and 3W, respectively. Immunoreactivity for p75(NTR) was observed on the surface of taste cells on and after P9. The results of the present study suggest that sensory structures in the laryngeal mucosa were developed on perinatal days to involve respiratory reflex, and that neurotrophin receptors may take part in the regulation and maintenance of sensory structures.

Localization of eNOS in the olfactory epithelium of the rat.

Nitric oxide (NO) is a free radical and produced from L-arginine by nitric oxide synthase (NOS). Since NO is recently suggested to be involved in olfactory perception, the expression of eNOS, an isoform of NOS, was examined in the rat olfactory epithelium. The activity of NADPH-diaphorase was also examined as a marker of NOS. In the dorsomedial region of the nasal cavity, intensely positive reactions for NADPH-diaphorase were observed in the entire cytoplasm of sensory cells (olfactory cells). By immunohistochemistry, intensely positive reactions for eNOS were also found in the dorsomedial region of the nasal cavity. These reactions were observed on the free border of the olfactory epithelium. By immunoelectron microscopy, positive reactions for eNOS were found in the cilia of olfactory cells. In addition, in situ hybridization analysis of the olfactory epithelium revealed the expression of eNOS mRNA in the olfactory cells. These results indicate the presence of eNOS in the olfactory cells of the rat, and differential expression of eNOS in the olfactory epithelium depending on the regions of the nasal cavity. In addition, NO produced by eNOS may be involved in olfactory perception in the cilia of olfactory cells.

Differences in tyrosine hydroxylase expression after short-term hypoxia, hypercapnia or hypercapnic hypoxia in rat carotid body.

In the carotid body (CB), it has been reported that the expressions of tyrosine hydroxylase (TH) mRNA and TH protein are enhanced by exposure to hypoxia. However, it is not known whether CO(2) affects the expression of TH in the CB. We examined the expression of TH mRNA and the immunoreactivity for TH in the CB of rats exposed to hypoxia (10% O(2)), hypercapnia (10% CO(2)) and hypercapnic hypoxia (10% O(2) and 10% CO(2)) for 2-24 h. The expression of TH mRNA in the CB was markedly enhanced in rats exposed to hypoxia for 4 h (6.6-fold), 6 h (6.0-fold) and 8 h (7.8-fold), and in rats exposed to hypercapnic hypoxia for 12 h (4.8-fold). The most intense TH immunoreactivity was observed in the CB from rats exposed to hypoxia for 12 and 24 h and to hypercapnic hypoxia for 24 h. The expressions of TH mRNA and the immunoreactivity for TH were not altered in the CB of rats exposed to hypercapnia. It is suggested that CO(2) does not affect TH expression in the CB, and that it inhibits hypoxia-enhanced TH expression.

Short-term hypoxia increases tyrosine hydroxylase immunoreactivity in rat carotid body.

Neurochemical and morphological changes in the carotid body are induced by chronic hypoxia, leading to regulation of ventilation. In this study, we examined the time courses of changes in immunohistochemical intensity for tyrosine hydroxylase (TH) and cellular volume of glomus cells in rats exposed to hypoxia (10% O(2)) for up to 24 hr. Grayscale intensity for TH immunofluorescence was significantly increased in rats exposed to hypoxia for 12, 18, and 24 hr compared with control rats (p<0.05). The transectional area of glomus cells was not significantly different between experimental groups. The TH fluorescence intensity of the glomus cells exhibited a strong negative correlation with the transectional area in control rats (Spearman's rho = -0.70). This correlation coefficient decreased with exposure time, and it was lowest for the rats exposed to hypoxia for 18 hr (rho = -0.18). The histogram of TH fluorescence intensity showed a single peak in control rats. The peaks were gradually shifted to the right and became less pronounced in hypoxia-exposed rats, suggesting that a hypoxia-induced increase in TH immunoreactivity occurred uniformly in glomus cells. In conclusion, this study demonstrates that short-term hypoxia induces an increase in TH protein expression in rat carotid body glomus cells.

Glutamate- and GABA-mediated neuron-satellite cell interaction in nodose ganglia as revealed by intracellular calcium imaging.

In the sensory ganglia, neurons are devoid of synaptic contacts, and ganglion neurons surrounded by one of glial cells, satellite cells. Recent studies suggest that neurons and satellite cells interact through neurotransmitters. In the present study, intracellular Ca(2+) ([Ca(2+)](i)) dynamics of neurons and satellite cells from one of viscerosensory ganglia, nodose ganglion (NG), were investigated by stimulation with glutamate and its agonist and/or the antagonist of the GABA(A) receptor bicuculline. In the specimens containing neurons with satellite cells, glutamate and a metabotropic glutamate receptor (mGluR) agonist t-ACPD evoked [Ca(2+)](i) increases in both neurons and surrounding satellite cells. Moreover, bicuculline also induced [Ca(2+)](i) increases in neurons and satellite cells. However, in the isolated neurons, bicuculline did not cause an increase in [Ca(2+)](i), suggesting that satellite cells are equipped with the ability to release GABA. In the neurons associated with satellite cells, the delay time until the onset of a response was shorter in the case of glutamate stimulation with bicuculline than that without bicuculline (107.3 +/- 93.4 vs. 231.8 +/- 97.0 s, p < 0.01). Furthermore, immunoreactivities for glutamate transporter, GLAST, and GABA transporter, GAT-3, were observed in both neurons and satellite cells of NG. In conclusion, the levels of [Ca(2+)](i) of NG neurons and surrounding satellite cells are increased by glutamate through at least mGluRs, and endogenous GABA modulates these responses; GABA inhibition is dependent on a close association between neurons and satellite cells. Such neuron-glia interaction in the nodose ganglion may regulate sensory information from visceral organs.

Localization of tissue transglutaminase (tTG) in kidney of ICR-derived glomerulonephritis (ICGN) mice.

ICR-derived glomerulonephritis (ICGN) mice are a known inbred strain with hereditary nephrotic syndrome and are considered a good animal model of human idiopathic nephrotic syndrome. ICGN mice show proteinuria at a young age, and hypoalbuminemia, hyperlipidemia, anemia and edema accompanies their symptoms with aging. In addition, ICGN mice develop severe anemia with the progression of renal fibrosis similar to human chronic kidney disease (CKD). Recently, tissue transglutaminase (tTG) has been shown to be related to the renal fibrosis in several animal models and CKD patients. In the present study, we investigated the relationship between the progression of renal fibrosis and the localization of tTG in the kidneys using histochemistry and image analysis. Male ICGN mice aged 26-43 weeks were used. They were divided into two groups of early and terminal stages of renal fibrosis, based on plasma levels of blood urea nitrogen (BUN). Normal ICR males aged 11 weeks were used as a control group. tTG was localized to the interstitium in the normal ICR mice. In the early stage of renal fibrosis, the localization of tTG increased in renal tubules showing luminal dilation, as well as in the interstitium; however, the amount of tubular and interstitial tTG decreased in the late stage. In the glomeruli, tTG-immunoreactivity decreased in the late stage of renal fibrosis, despite the progression of glomerular sclerosis. The results suggest that epsilon(gamma-glutamyl) lysine cross-linking is not directly related to the progression of renal fibrosis in ICGN mice.

Immunohistochemical distribution of inwardly rectifying K+ channels in the medulla oblongata of the rat.

The inwardly rectifying K+ channels, Kir1.1, Kir2.3 and Kir4.1-Kir5.1, are the candidate chemosensory molecules for CO2/H+. We determined the mRNA expression and immunohistochemical localization of these channels in the medulla oblongata of the rat. RT-PCR analysis revealed mRNAs of Kir1.1, Kir2.3, Kir4.1 and Kir5.1 were detected in the medulla. The immunoreactivities for Kir1.1, Kir2.3, Kir4.1, and Kir5.1 were observed in the medulla, and immunolabeling pattern was varied by the subunit. Immunoreactivities for Kir1.1 and Kir2.3 were observed in the nerve cell bodies and glial cells both in the chemosensory areas [nucleus tractus solitarius (NTS), nucleus raphe obscurus (RO), pre-Bötzinger complex (PreBötC)] and non-chemosensory area [hypoglossal nucleus (XII), inferior olive nucleus (IO)]. Kir4.1 immunoreactivity was observed in the glial cells and neuropil, especially in XII and IO. Kir5.1 immunoreactivity was observed in the nerve cell bodies in the XII, RO, and PreBötC, but not in the NTS or IO. In the NTS, a dense network of varicose nerve fibers showed immunoreactivity for Kir5.1. Our findings suggest that Kir channels may not act specific to the central chemoreception, but regulate the ionic properties of cellular membranes in various neurons and glial cells.

Expression of Fos protein in brainstem after application of l-menthol to the rat nasal mucosa.

There are two functional pathways for the nasotrigeminal reflex: the spinal nucleus of trigeminal nerve (SPV) to the Kölliker-Fuse (KF) nucleus and the nucleus of solitary tract (NTS) to the lateral parabrachial nucleus (PBl). Although stimulation of the nasal mucosa by cool temperature induces respiratory depression, it is still unknown whether these nuclei are activated. In the present study, we examined the expression of Fos protein in rat brainstem neurons after nasal application of l-menthol, which is known to activate cold-sensitive nasal receptors. Application of l-menthol, but not paraffin oil, decreased the respiratory rate from 99.7+/-15.6 to 78.5+/-7.3 min(-1). Furthermore, a significantly higher density of Fos-immunoreactive cells was observed in the SPV and KF in the l-menthol rats than in the controls. In the SPV, the density of Fos-immunoreactive cells was highest at approximately 0.5mm rostral to the obex in both the l-menthol (48.5+/-11.5 cells/section) and paraffin oil (26.0+/-9.6 cells/section) groups. In the KF, the mean density of Fos-immunoreactive cells was highest at approximately 5.0mm rostral to the obex in both groups (l-menthol: 67.8+/-14.0 cells/section, control: 41.0+/-12.7 cells/section). The present study suggests that the SPV-KF pathway is important for the cold-induced respiratory depression.

Erythropoietin-producing cells in the liver of ICR-derived glomerulonephritis (ICGN) mice.

The ICR-derived glomerulonephritis (ICGN) mouse is an appropriate model for anemia associated with chronic renal disorder (CRD). Insufficient renal production of erythropoietin (EPO) induces the anemia associated with CRD. EPO mRNA is expressed in both kidneys and liver of progressing-stage ICGN mice. Hypoxic stimulation induced the EPO mRNA expression in the liver as well as in the kidneys of ICGN mice. The localization of EPO-producing cells in the liver remains controversial. Present study using an amplified in situ hybridization technique identified that nonparenchymal cells were the source of hepatic EPO production in ICGN mice under both normoxia and hypoxia.

Dysfunction of erythropoietin-producing interstitial cells in the kidneys of ICR-derived glomerulonephritis (ICGN) mice.

Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.

Effect of human erythropoietin (hEPO) treatment on anemia in ICR-derived glomerulonephritis (ICGN) mice.

ICR-derived glomerulonephritis (ICGN) mice are a novel inbred strain with hereditary nephrotic syndrome and are thus considered a good animal model of human idiopathic nephrotic syndrome. In the present study, we investigated the effect to erythrocyte production by human erythropoietin (hEPO) treatment in ICGN mice during the early nephrotic stage. Erythrocyte count, hemoglobin concentration and hematocrit value in hEPO-treated (5 U/body/day, for 5 days) ICGN mice were recovered to the levels found in normal ICR mice. In addition, there was no correlation between plasma creatinine level, a marker of renal function, and erythrocyte count after hEPO treatment. Therefore, anemia in ICGN mice may be caused by decreased production of EPO in the kidney following progressive parenchymal damage.

Improvement of anemia associated with chronic renal failure by recombinant human erythropoietin treatment in ICR-derived glomerulonephritis (ICGN) mice.

The ICR-derived glomerulonephritis (ICGN) mouse, a novel inbred mouse strain with a hereditary nephrotic syndrome, develops severe anemia associated with chronic renal failure. To reveal the pathogenic mechanism of anemia in ICGN mice, we subcutaneously administered recombinant human erythropoietin (rhEPO; 5 IU/mouse/day) or saline for 5 days to ICGN mice. In terminal-stage ICGN mice with severe anemia, rhEPO significantly increased hematocrit (Ht), red blood cells (RBC) and hemoglobin levels. Endogenous EPO levels in peripheral blood were reduced by rhEPO injection. No histopathological changes in bone marrow and kidneys were induced by rhEPO injection. Insufficiency of EPO may cause anemia in ICGN mice.

Anemia with chronic renal disorder and disrupted metabolism of erythropoietin in ICR-derived glomerulonephritis (ICGN) mice.

The ICR-derived glomerulonephritis (ICGN) mouse, a new inbred mouse strain with a hereditary nephrotic syndrome, is considered to be a good model of human idiopathic nephrotic syndrome and notably exhibits proteinuria and hypoproteinemia from the neonatal stage. In chronic renal disorder (CRD), anemia is a major subsequent symptom (renal anemia). The precise cause of renal anemia remains unclear, primarily owing to the lack of appropriate spontaneous animal models for CRD. To establish adequate animal models for anemia with CRD, we examined the hematological-biochemical properties and histopathological characteristics. With the deterioration of renal function, ICGN mice developed a normochromic and normocytic anemia, and exhibited normochromic and microcytic at the terminal stage. The expression of erythropoietin (EPO) mRNA both in the kidneys and liver and the EPO leak into the urine were observed in ICGN mice, indicating a disrupted metabolism of EPO in ICGN mice. In addition, a lack of iron induced by the hemolysis in the spleen and the leak of transferrin into urine as proteinuria aggravated the anemic condition. In conclusion, the ICGN mouse is a good model for anemia with CRD.

Changes in the localization of type I, III and IV collagen mRNAs in the kidneys of hereditary nephritic (ICGN) mice with renal fibrosis.

Renal fibrotic change, extreme accumulation of extracellular matrix (ECM) components in glomeruli and tubulointerstitum, is one of the characteristic features of ICR-derived glomerulonephritis (ICGN) mice. Decreased degradation of ECMs by matrixmetalloproteinases was demonstrated in kidneys of ICGN mice. To determine the balance between production and degradation of ECMs in kidneys of ICGN mice, we examined expression of mRNAs of ECMs in those. To demonstrate the localization of type I, III and IV collagen mRNAs in kidney sections of ICGN and control ICR mice, in situ hybridization using digoxigenin-labeled oligonucleotide antisense probes for procollagen-alpha(1) (I), -alpha(1) (III) and -alpha(1) (IV) mRNAs, respectively, was performed. Negative or trace expressions of type I and III collagen mRNAs were observed in the kidneys of control mice, but stronger expressions of those were seen in glomeruli and injured renal tubules of the kidneys of ICGN mice. Moderate expression of type IV collagen mRNA was demonstrated in a part of glomeruli and renal tubules of both control and ICGN mice, and no remarkable difference was seen between them. Severe renal fibrosis, extreme accumulation of interstitial type I and III collagens is caused by increased production and decreased degradation in the kidneys of ICGN mice. Thus, the profiles of metabolism between interstitial and membranous collagens may be different in the kidneys of ICGN mice, and excessive production of interstitial collagens may be the dominant cause of renal disease in them.