Expression of an inwardly rectifying K+ channel, Kir4.1, in satellite cells of rat cochlear ganglia

Satellite cellsare glial cells wrapped around somata of sensory and autonomic ganglionneurons. Neither their functional roles nor electrical properties havebeen fully clarified so far. Using immunohistochemistry, we found thatinwardly rectifying K⁺ channelsubunit Kir4.1 (also called Kir1.2 orK_AB-2) was expressed prominently in the satellite cells of cochlear ganglia. The Kir4.1 immunoreactivity was localized specifically at the myelin sheaths ofsatellite cells wrapping the somata of the ganglion neurons. Developmental expression of Kir4.1 in satellite cells paralleled development of the action potential in the auditory nerve. These results suggest that this channel in satellite cells may be responsible for the regulation of K⁺ extrudedfrom the ganglion neurons during excitation.

     

Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations

Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.     

Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1

BACE (β-site amyloid precursor protein cleaving enzyme, β-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of β-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, γ-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans -Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.     

Autophagy–physiology and pathophysiology

“Autophagy” is a highly conserved pathway for degradation, by which wasted intracellular macromolecules are delivered to lysosomes, where they are degraded into biologically active monomers such as amino acids that are subsequently re-used to maintain cellular metabolic turnover and homeostasis. Recent genetic studies have shown that mice lacking an autophagy-related gene (Atg5 or Atg7) cannot survive longer than 12 h after birth because of nutrient shortage. Moreover, tissue-specific impairment of autophagy in central nervous system tissue causes massive loss of neurons, resulting in neurodegeneration, while impaired autophagy in liver tissue causes accumulation of wasted organelles, leading to hepatomegaly. Although autophagy generally prevents cell death, our recent study using conditional Atg7-deficient mice in CNS tissue has demonstrated the presence of autophagic neuron death in the hippocampus after neonatal hypoxic/ischemic brain injury. Thus, recent genetic studies have shown that autophagy is involved in various cellular functions. In this review, we introduce physiological and pathophysiological roles of autophagy.     

Effect of low root temperature on hydraulic conductivity of rice plants and the possible role of aquaporins

The role of root temperature T(R) in regulating the water-uptake capability of rice roots and the possible relationship with aquaporins were investigated. The root hydraulic conductivity Lp(r) decreased with decreasing T(R) in a measured temperature range between 10 degrees C and 35 degrees C. A single break point (T(RC) = 15 degrees C) was detected in the Arrhenius plot for steady-state Lp(r). The temperature dependency of Lp(r) represented by activation energy was low (28 kJ mol(-1)) above T(RC), but the value is slightly higher than that for the water viscosity. Addition of an aquaporin inhibitor, HgCl(2), into root medium reduced osmotic exudation by 97% at 25 degrees C, signifying that aquaporins play a major role in regulating water uptake. Below T(RC), Lp(r) declined precipitously with decreasing T(R) (E(a) = 204 kJ mol(-1)). When T(R) is higher than T(RC), the transient time for reaching the steady-state of Lp(r) after the immediate change in T(R) (from 25 degrees C) was estimated as 10 min, while it was prolonged up to 2-3 h when T(R) < T(RC). The Lp(r) was completely recovered to the initial levels when T(R) was returned back to 25 degrees C. Immunoblot analysis using specific antibodies for the major aquaporin members of PIPs and TIPs in rice roots revealed that there were no significant changes in the abundance of aquaporins during 5 h of low temperature treatment. Considering this result and the significant inhibition of water-uptake by the aquaporin inhibitor, we hypothesize that the decrease in Lp(r) when T(R) < T(RC) was regulated by the activity of aquaporins rather than their abundance.     

Angiotensin II receptor antagonism reverts the selective cardiac BNP upregulation and secretion observed in myocarditis

The cardiac natriuretic peptides atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) are discoordinately regulated in myocardial inflammation associated with acute allograft rejection in humans and during in vitro exposure of cardiocyte cultures to some proinflammatory cytokines. We used experimental autoimmune myocarditis (EAM) to determine whether the discoordinate regulation of ANF and BNP was specific to the situations above or was generally associated with other types of myocardial inflammation. The dependency of this process to angiotensin signaling was also determined, given that previous work demonstrated beneficial effects of the angiotensin receptor blocker olmesartan in myocarditis. Histopathological changes, plasma and cardiac ANF, BNP, and selected cytokines gene expression as well as plasma cytokine levels using a cytokine array were determined in EAM, angiotensin receptor blocker-treated, and control rats. It was found that EAM specifically increases BNP but not ANF circulating levels, thus mimicking the findings in acute cardiac allograft rejection and the effect of some proinflammatory cytokines on cardiocyte cultures in vitro. Plasma cytokine array and real-time PCR revealed that lipopolysaccharide-induced CXC chemokine, monocyte chemotactic protein-1, and tissue inhibitor of metalloproteinase-1 were increased in plasma and in the myocardium of EAM rats. Olmesartan treatment reversed virtually all neuroendocrine and histopathological cardiac changes induced by EAM, thus providing a mechanistic insight into this phenomenon. It is concluded that the inflammatory process contributes specific cytokines, leading to the disregulation of cardiac ANF and BNP production observed during myocardial inflammation, and that this process is angiotensin receptor 1 dependent.     

Calcitonin gene-related peptide (CGRP)-immunoreactive nerve plexuses in the renal pelvis and ureter of rats

Summary The distribution of calcitonin gene-related peptide-immunoreactive nerve fibers in the renal pelvis and ureter was examined by immunohistochemistry using whole-mount preparations and cryostat sections. The patterns of innervation were contrasted between the pelvis and ureter; the immunoreactive nerve fibers in the pelvis ran parallel to the long axis of each of the circular and longitudinal muscle layers, causing a lattice-like appearance of the nerve fibers. In the ureter, the immunoreactive fibers were accumulated in the subepithelial region and the longitudinal muscle. In both the pelvis and ureter, a portion of the nerve fibers of smaller caliber showed a swollen or beaded structure; they were located in the musculature and beneath the epithelium extending for considerable distances. Ligation of the ureter caused a marked decrease in the immunoreactive nerves in the pelvis and the proximal portion of the ureter, suggesting that the axonal flow in the calcitonin gene-related peptide-containing neurons of the ureter runs towards the pelvis.     

Immunocytochemical localization of prorenin, renin, and cathepsins B, H, and L in juxtaglomerular cells of rat kidney

To examine the correlation of localization of prorenin, renin, and cathepsins B, H, and L, immunocytochemistry was applied to rat renal tissue, using a sequence-specific anti-body (anti-prorenin) that recognizes the COOH terminus of the rat renin prosegment. In serial semi-thin sections, immunodeposits for prorenin, renin, and cathepsins B, H, and L were localized in the same juxtaglomerular (JG) cells. Immunodeposits for renin were detected throughout the cytoplasm of the cells, whereas those for prorenin were detected in the perinuclear region. Immunoreactivity for cathepsin B was stronger than that for cathepsins H and L. By electron microscopy, prorenin was localized in small (immature) granules but not in large mature granules, whereas renin was localized mainly in mature granules. In serial thin sections, prorenin, renin, and cathepsin B were colocalized in the same immature granules containing heterogeneously dense material (intermediate granules). By double immunostaining, co-localization of renin with cathepsins B, H, or L was demonstrated in mature granules. The results suggest the possibility that processing of prorenin to renin occurs in immature granules of rat JG cells, and cathepsin B detected in JG cells may be a major candidate for the maturation of renin.