Koichiro Omori

Vascular endothelial growth factor expression in choroidal neovascularization in rats

Background: The pathogenesis of choroidal neovascularization is largely unknown. We investigated vascular endothelial growth factor (VEGF) expression in laser-induced choroidal neovascularization (CNV) in rats.Methods: Intense krypton laser photocoagulation was applied to the posterior poles of the eyes of pigmented rats to induce CNV, which was confirmed by fluorescein angiography and histopathology. The eyeballs were enucleated 1, 3, 7, 14 and 28 days after laser photocoagulation. Cryostat sections were prepared for immunofluorescence staining using anti-VEGF and macrophage marker (ED1) antibodies. The posterior segments of eyeballs pooled from photocoagulated and control rats were submitted for immunoprecipitation and immunoblotting by the anti-VEGF antibody, and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VEGF mRNA.Results: Very weak immunoreactivity for anti-VEGF antibody was found in the ganglion cell layer, inner nuclear layer, and retinal pigment epithelium (RPE) in the normal retina. In the development of CNV, strong positive staining for anti-VEGF antibody was found in photocoagulated areas in the subretinal space and choroid. Double immunofluorescence staining showed that many cells in lasered lesions were positive both for anti-VEGF and macrophage marker ED1 antibody staining in the early stage of this model. Immunoblots showed a positive band for the VEGF molecule in treated but not control animals. RT-PCR results demonstrated upregulation of VEGF transcripts in the CNV model compared with normal animals.Conclusions: Our findings showed the upregulation of VEGF expression in experimentally induced CNV, where it may be involved in promoting choroidal angiogenesis. Macrophages may be one of the main sources of VEGF in the early stage of the disease.

Quantitative immunocytochemical localization of Na+,K+-ATPase alpha-subunit in the lateral wall of rat cochlear duct.

Ultrastructural localization of the alpha-subunit of Na+,K+-ATPase on the lateral wall of rat cochlear duct was investigated quantitatively by the protein A-gold method, using affinity-purified antibody against the alpha-subunit of rat kidney Na+,K+-ATPase. In the stria vascularis, gold particles were sparse over the endolymphatic luminal surface of the marginal cells but were numerous over the basolateral membrane. The labeling density of the basolateral membrane was almost equal to that of the same domain of the distal tubule cells of kidney. The intermediate cells were studded with a large number of gold particles on the plasma membrane domain facing the basolateral domain of the marginal cells. On the luminal surfaces of the other epithelial cells, including those of Reissners membrane, no significant amount of gold particles was found. Many gold particles were localized on all the plasma membranes of the spiral prominence stromal cells and on the intracellular membrane domain of the external sulcus cells.

Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells.

We investigated quantitatively the ultrastructural localization of the alpha-subunit of Na+,K(+)-ATPase in rat retinal pigment epithelial cells by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha- and alpha(+)-subunits of Na+,K(+)-ATPase in the whole retina [the sensory retina plus retinal pigment epithelium (RPE)]. Rat eyes were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde and embedded in Lowicryl K4M. Ultra-thin sections were incubated with affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase and subsequently with protein A-gold complex. Light microscopy with a silver enhancement procedure revealed Na+,K(+)-ATPase localized to both the apical and the basal plasma membrane domains of the RPE. Quantitative immunocytochemical analysis by electron microscopy showed a higher density of gold particles on the apical surface than on the basolateral one. Microvilli are so well developed on the apical surface of the RPE that the apical surface profile is much longer than the basolateral one. This means that Na+,K(+)-ATPase is mainly located on the apical surface of the RPE cells.

The chloride channel ClC-2 contributes to the inwardly rectifying Cl- conductance in cultured porcine choroid plexus epithelial cells.

1 The contribution of ClC‐2 protein to the inwardly rectifying Cl− conductance in cultured porcine choroid plexus epithelial cells was investigated using Western analysis and whole‐cell current recordings. 2 Inwardly rectifying currents were elicited by hyperpolarizing voltage at a potential more negative than −50 mV in the presence of intracellular protein kinase A (PKA). The relative halide selectivity estimated from the shift in the reversal potential (Erev) was I− > Br− > Cl− > F−. 3 Extracellular vasoactive intestinal peptide (VIP) activated the same currents in a dose‐dependent manner with a half‐maximal concentration of 167·3 nM. H‐89 (a PKA inhibitor) interfered with the current activation by VIP. 4 The Cl− channel was inhibited by external Cd2+, Ba2+or H+, but only weakly inhibited by known Cl− channel blockers including glibenclamide, NPPB, DIDS and anthracene‐9‐carboxylic acid (9AC). 5 A specific antibody to ClC‐2 detected a 79 kDa protein in porcine choroid plexus cells, which was reduced in cells treated with antisense oligodeoxynucleotide for ClC‐2. Both PKA and VIP failed to activate the inwardly rectifying Cl− currents in cells transfected with the antisense oligodeoxynucleotide, while they activated the currents in cells transfected with GFP alone or the control oligodeoxynucleotide randomized from antisense oligonucleotide. 6 It is concluded that ClC‐2 protein contributes to the inwardly rectifying Cl− conductance in porcine choroid plexus epithelial cells.

GABAC receptor agonist suppressed ammonia‐induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level

Ammonia‐induced apoptosis and its prevention by GABAC receptor stimulation were examined using primary cultured rat hippocampal neurons. Ammonia (0.5–5 mm NH4Cl) dose‐dependently induced apoptosis in pyramidal cell‐like neurons as assayed by double staining with Hoechst 33258 and anti‐neurofilament antibody. A GABAC receptor agonist, cis‐4‐aminocrotonic acid (CACA, 200 µm), but not GABAA and GABAB receptor agonists, muscimol (10 µm) and baclofen (50 µm), respectively, inhibited the ammonia (2 mm)‐induced apoptosis, and this inhibition was abolished by a GABAC receptor antagonist (1,2,5,6‐tetrahydropyridin‐4‐yl)methylphosphinic acid (TPMPA, 15 µm). Expression of all three GABAC receptor subunits was demonstrated in the cultured neurons by RT‐PCR. The ammonia‐treatment also activated caspases‐3 and ‐9 as observed in immunocytochemistry for PARP p85 and western blot. Such activation of the caspases was again inhibited by CACA in a TPMPA‐sensitive manner. The anti‐apoptotic effect of CACA was blocked by inhibitors for MAP kinase kinase and cAMP‐dependent protein kinase, PD98059 (20 µm) and KT5720 (1 µm), suggesting possible involvement of an upstream pro‐apoptotic protein, BAD. Levels of phospho‐BAD (Ser112 and Ser155) were decreased by the ammonia‐treatment and restored by coadministration of CACA. These findings suggest that GABAC receptor stimulation protects hippocampal pyramidal neurons from ammonia‐induced apoptosis by restoring Ser112‐ and Ser155‐phospho‐BAD levels.

Inhibition of cardiac delayed rectifier K+ currents by an antisense oligodeoxynucleotide against IsK (minK) and over-expression of IsK mutant D77N in neonatal mouse hearts.

Abstract. The IsK (minK or KCNE1) protein is known to co-assemble with the KvLQT1 (KCNQ1) protein to form a channel underlying the slowly activating delayed rectifier K+ current (IKs). Controversy remains as to whether the IsK protein assembles with ERG (the ether-a-go-go-related gene) products to form or modulate the channel underlying the rapidly activating delayed rectifier K+ current (IKr). We investigated the effects of antisense oligodeoxynucleotides (AS-ODN) against IsK and its mutant D77N [which underlies a form of long QT syndrome (LQT5) in humans] on the delayed rectifier K+ current (IK) of neonatal mouse ventricular myocytes in primary culture. Patch-clamp experiments on these cells showed that IK consists of IKs and IKr. IK was not recorded from ventricular cells transfected with AS-ODN, while it was recorded from cells transfected with the corresponding sense oligodeoxynucleotides (S-ODN). IK was not recorded from cells transfected with the D77N mutant, and the action potential duration was much longer than in cells transfected with wild-type IsK. Furthermore, HERG could not induce currents in COS-1 cells co-expressed with the D77N mutant and HERG (the human form of ERG). These results indicate that the IsK protein associates with both KvLQT1 and ERG products to modulate IKr and IKs in cardiac myocytes.

Neutralization of aspartate residues in the murine inwardly rectifying K+ channel IRK1 affects the substate behaviour in Mg2+ block

1 To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single‐channel currents through inwardly rectifying K+ (IRK1) channels were studied. 2 cDNA encoding a functional murine IRK1 channel was transfected into COS‐1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48‐72 h. 3 Intracellular Mg2+ at micromolar concentrations induced sublevels in the outward current at one‐third and two‐thirds of the unitary amplitude seen in wild‐type channels. Replacing Asp 172 with Asn (D172N) and Gln (D172Q) abolished these sublevels, i.e. the channel showed only the fully open and fully blocked states. 4 Both mutations reduced the Mg2+ sensitivity of the channel at 2 μM Mg2+. However, the Mg2+ sensitivity did not differ significantly at higher concentrations (10 μM) and voltages (+70 mV). 4 Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behaviour. 6 Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two‐thirds amplitude, while those from tetramers with three D172N mutant subunits showed no sublevels. 7 These findings suggest that differences in Mg2+ binding patterns lead to different conductive states in a single‐barrelled channel.

Voltage-dependent gating and block by internal spermine of the murine inwardly rectifying K+ channel, Kir2.1.

The mechanism of inward rectification was investigated by recording single‐channel currents through an inwardly rectifying K+ channel (Kir2.1). cDNA encoding a wild‐type (WT) channel, a mutant replacing Asp 172 with Asn (D172N), and a tandem tetramer WT‐(D172N)2‐WT, was transfected into COS‐1 cells using the liposome method, and after 48–72 h single‐channel currents were recorded in the inside‐out configuration at 150 mm internal and external K+. Steady‐state open probability of outward currents decreased with larger depolarizations. The activation curve was fitted with a single Boltzmann equation. The voltages of half‐activation in the absence of spermine were +35.9 mV (WT), +55.0 mV (WT‐(D172N)2‐WT) and +76.7 mV (D172N). Open‐time and zero‐current‐time histograms were constructed. The open‐time histogram was fitted with a single exponential function. Two exponential functions were necessary to fit the closed‐time histogram. In each channel, internal spermine at a concentration of 1–100 nm reduced the open time of the outward currents in a concentration‐dependent manner and produced one blocked state without affecting the inward currents, suggesting that spermine acts as an open channel blocker. The normalized steady‐state open probability‐spermine concentration curve was fitted by saturation kinetics with a Hill coefficient of 1. On the assumption of the linear sequential state model, the unblock and blocking rates were estimated in each channel. Unblock rates depended on the number of D172N mutant subunits, but blocking rates did not. The results suggest that closing gates work independently of the spermine block and D172 is involved in both intrinsic gating and the spermine block.

Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells.

Lysyl oxidase, an extracellular amine oxidase, controls the maturation of collagen and elastin. We examined the regulation of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium (RPE) cells in relation to the changes in subretinal fluid transport and phenotype of RPE cells. The level of the mRNA in cells grown on microporous membranes was markedly increased by application of hyperosmotic mannitol solution on the apical side (191% of control), implying that RPE cells express more lysyl oxidase in the condition which may cause the accumulation of subretinal fluid. Platelet-derived growth factor increased the mRNA level in subconfluent cells in culture (137% of control) and basic fibroblast growth factor decreased it (79% of control). In addition, exposure of cells to retinoic acid alone or in combination with dibutyryl cAMP for 22 days markedly decreased the level of lysyl oxidase mRNA (52 or 35% of control) while increasing the level of mRNA of N-acetylglucosaminidase (NAG), a marker enzyme for lysosomes (162 or 142% of control). Moreover, the level of lysyl oxidase mRNA in cells grown on microporous membranes was lower than that in cells grown on plastic dishes, while the level of NAG mRNA in the former cells was higher than that in the latter. Taken together, the expression of lysyl oxidase seemed to increase during proliferation of RPE cells and decrease toward differentiation. beta-Aminopropionitrile, an inhibitor of lysyl oxidase, significantly inhibited the contraction of collagen gels by fetal calf serum, suggesting that lysyl oxidase may be involved in pathogenesis caused by RPE cells.

Inwardly rectifying potassium channels expressed by gene transfection into the green Monkey kidney cell line COS‐1.

1. cDNA encoding a functional inwardly rectifying K+ (IRK1) channel was transfected into COS‐1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48‐72 h. 2. Transfected cells showed inward rectification under whole‐cell recording. The unitary current‐voltage relationships in the inside‐out configuration were almost linear in the absence of internal Mg2+ and polyamines, and the channel conductance averaged 34.1 +/‐ 2.0 pS (n = 15) at 23‐26 degrees C. 3. Internal Mg2+ (2‐10 microM) induced sublevels in the outward current with one‐third and two‐thirds of the unitary amplitude as in native channels. 4. To determine the subunit stoichiometry, we constructed tandem multimeric cDNAs consisting of the coding sequences of the IRK1 gene linked in a head‐to‐tail fashion. Cells transfected with tandem homomultimers up to octamers showed similar inwardly rectifying K+ channels. 5. A mutation (E138Q) eliminated the ionic conductance of the channel. Channels expressed by dimeric constructs containing a single mutant have a conductance ranging between 5 and 35 pS. 6. The E138Q mutant cotransfected with a wild‐type dimeric, trimeric or tetrameric construct did not alter the channel conductance. The results do not support the notion that IRK1 channel proteins consist of four subunits.