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.

Quantitative immunocytochemical localization of Na+,K+-ATPase in rat ciliary epithelial cells.

Ultrastructural localization of Na+,K+-ATPase in rat ciliary epithelium was investigated quantitatively by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of Na+,K+-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha-subunit of Na+,K+-ATPase in the ciliary body. Gold particles were found mainly on the basolateral surfaces of both the pigmented epithelial (PE) and nonpigmented epithelial (NPE) cells with an approximately twofold higher labeling density in the PE cells. A few gold particles were also found on the apical and ciliary channel surfaces of the PE cells, whereas no significant binding was found on the apical surfaces of the NPE cells. The basolateral surfaces of PE and NPE cells are markedly infolded and are much greater in area than the apical surfaces. This means that Na+,K+-ATPase is almost exclusively located on the basolateral surfaces of both the NPE and PE cells. We suggest that the Na+,K+-ATPase of both the NPE and PE cells play an important role in the formation of aqueous humor.

Ultrastructural localization of Na+, K(+)-ATPase in the exorbital lacrimal gland of rat.

Ultrastructural localization of Na+, K(+)-ATPase in the exorbital lacrimal gland of rat was investigated quantitatively by protein A-gold technique. Na+, K(+)-ATPase was purified from the rat kidney, and anti-holo Na+, K(+)-ATPase antibody was obtained from the rabbit by injecting the purified enzyme. A specific antibody against the alpha-subunit of Na+, K(+)-ATPase was affinity purified. Immunoblot analysis revealed that the antibody bound specifically to the alpha-subunit of Na+, K(+)-ATPase of the lacrimal gland. Rats were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde, and the lacrimal glands were embedded in LR White resin. Ultrathin sections were incubated with affinity purified antibody against the alpha-subunit of Na+, K(+)-ATPase, and then with protein A-gold complex. The sections were observed under an electron microscope. Light microscopy with silver enhancement procedure revealed that Na+, K(+)-ATPase was located mainly on the basal region of the cells of intralobular and interlobular ducts. Quantitative immunoelectron microscopic analysis showed that gold particles were found on the basolateral surfaces of the interlobular and intralobular ducts cells and on the basolateral surface of the acinar cells, whereas no significant binding was observed on any part of the apical surfaces of these cells. Labeling density of gold particles was highest on the basolateral surface of the interlobular duct cells, secondarily highest on the basolateral surface of the intralobular duct cells, and lowest on the basolateral surface of the acinar cells. The distribution pattern of Na+, K(+)-ATPase in the acinar cells and the duct cells suggest that this enzyme may play an important role in primary secretion and in determining the composition of electrolytes in the final secretion, respectively.

[Ocular manifestations in a case of infantile cystinosis].

A case of infantile cystinosis was reported. The diagnosis of cystinosis was made by the renal Fanconi syndrome and the ocular findings. The patient showed typical corneal and fundus changes associated with cystinosis. Corneal crystals and fundus pigmentary change were found early in life. The deposition of corneal crystals increased in the course of the disease, especially in the nasal and the temporal sides close to the limbus. By specular microscopy, the corneal crystals were needle-shaped and were larger and more numerous in the superficial layer of the stroma. The same shape of crystals ere found in the corneal endothelium, and the size of endothelial cells was markedly increased. There were also small crystals on the surface of the iris. The entire fundus showed a mottled appearance characterized by mixture of fine granular pigmentation and depigmentation associated with patchy depigmentation in the periphery. On the anterior surface of the retina, a glistening crystal appeared. As the crystal deposition in ocular tissues increases, even after successful peritoneal dialysis, eventual impairment of visual function may be expected.

[Interferon-associated retinopathy].

During interferon (IFN) treatment for chronic hepatitis C, retinopathy develops in about one half of the patients. This "IFN-associated retinopathy" is characterized by cotton-wool spots (ischemic lesions) and superficial hemorrhage around the optic disc. It usually occurs 1 to 3 months after initiation of IFN therapy. Most of the patients with retinopathy have no symptoms, while some complain of impairment of visual acuity or flying specks. IFN-associated retinopathy tends to improve without any ophthalmic treatment and IFN therapy can be continued under careful observation. A complication of diabetes mellitus or a decrease in platelet count during IFN treatment may aggravate IFN-associated retinopathy. We emphasize that careful ophthalmologic follow-up is needed for all patients under IFN therapy.

A new resuscitation fluid "stabilized hemoglobin" preparation and characteristics.

A new oxygen carrier for use as a blood substitute was prepared and characterized in vitro. Pyridoxylated hemoglobin, which was obtained by the reaction of human hemoglobin with pyridoxal-5-phosphate, was modified by alpha-carboxymethyl, omega-carboxymethoxyl polyoxyethylene (POE) of the molecular weight 3600 daltons. In order to eliminate viruses and nucleic acids possibly contaminated, the hemoglobin solution was purified by ultrafiltration with a membrane of the nominal molecular weight limit 300 Kdaltons. Furthermore POE conjugated pyridoxylated hemoglobin was treated with 20% ethanol to inactivate viruses. A concentration of hemoglobin, which is incorporated in the conjugate, of the final product was fixed at 6% to make normovolemic exchange transfusion possible. In consideration of the stability during transporting and storage, lyophilized product was selected as a final form ("Stabilized Hemoglobin"). "Stabilized hemoglobin" could be stored in a refrigerator over one year within the acceptable methemoglobin increase. (15%) Viscosity of Stabilized Hemoglobin solution was determined at 2.4 centipoise and is almost half of whole blood and therefore this will be useful not only in resuscitation but also in improvement of microcirculation.

Immunocytochemical analyses of distributions of Na, K-ATPase and GLUT1, insulin and transferrin receptors in the developing retinal pigment epithelial cells.

The retinal pigment epithelium (RPE) is unique in that Na,K-ATPase is predominantly localized on its apical surface. We studied the distributions of Na,K-ATPase and glucose transporter GLUT1, insulin and transferrin receptors in developing rat RPE cells immunocytochemically. Na,K-ATPase, first detected in 17-day-old embryonic eyes, was already distributed predominantly on the apical surface. This reversed distribution of Na,K-ATPase was maintained throughout their life. Insulin receptor and transferrin receptor were distributed exclusively on the basolateral surface. By quantitative immunogold electron microscopic technique we found that glucose transporter GLUT1 is distributed almost equal in amount on both the apical and basolateral surfaces of RPE cells, thus presumably constructing an efficient pathway for glucose transport from the choriocapillaries to the neural retina through the blood-retinal barrier. These results suggest that in the RPE cells the intrinsic basolateral plasma membrane proteins are sorted out at least in three different ways.