Rat retinal progenitor cells and a retinal pigment epithelial factor.

Retinal progenitor cells were isolated from explants of neonatal rat retinas and characterized by transmission electron microscopy and reverse transcriptase-polymerase chain reaction and by their response to an isolated retinal pigment epithelial cell cell factor. The isolated progenitor cells demonstrated nuclei with abundant euchromatin typical of progenitor cells and showed the presence of nestin and opsin message. A protein ( approximately 67 kDa) isolated from conditioned media of cultured rat RPE cells promoted the survival of isolated retinal progenitor cells.

Effect of temperature on apical membrane remodeling in ADH-stimulated toad urinary bladders.

Pretreatment and removal of vasopressin (ADH) in toad urinary bladder renal model tissues induces endocytosis at 25 degrees C. The objective of the current study is to determine if apical membrane remodeling, as well as transepithelial water flow, can be affected by lowering the temperature to 15 degrees C. Control toad urinary bladders in the presence of an osmotic gradient at either 25 degrees C or 15 degrees C when visualized by scanning electron microscopy (SEM) show a typical apical membrane surface with no apparent surface differences. ADH-treated tissues following 15-min stimulation at 25 degrees C or 15 degrees C revealed a propagation of apical microvilli on their surface membranes. After 15 min following removal of ADH, bladder tissues at 25 degrees C or 15 degrees C showed surface invaginations involving over 44% and 80% of granular cells, respectively. The rate of water flow in tissues at 15 degrees C remained elevated compared to tissues held at 25 degrees C. This was consistent with the observation that ADH-stimulated tissues following washout at 15 degrees C still had marked apical membrane surface involvement. However, at 30 min and 60 min postwashout, ADH-stimulated tissues at 15 degrees C recovered considerably, with a reduction in the number of shallow apical membrane invaginations involving fewer than 33% and 20% of granular cells respectively. This may indicate that the membrane undergoes continuous remodeling even at cold temperature conditions but with a different half-time. Control bladder tissues subjected to transmission electron microscopy (TEM) reveal a dense cytoplasmic profile with a scattered distribution of secretory granules, rough ER cisternae, mitochondria, and little or no vacuolation. In contrast, ADH-stimulated bladder tissues displayed a vacuolated cytoplasm, expanded rough ER cisternae, and ruffled basolateral membranes. These observations suggest that the apical membrane undergoes considerable reorganization following cessation of hormone action and that lowering the temperature reduces the rate of membrane remodeling and thus may provide a means to monitor the processes of endocytosis and the mechanisms responsible for water channel retrieval.

Methionine-enkephalin-Arg-Phe immunoreactivity in heart tissue.

Previous findings of enkephalins in cardiac tissue led us to investigate enkephalin distribution in animal models used for cardiovascular research. Canine cardiac methionine-enkephalin (ME) concentrations are low and evenly distributed between atria (4.2 +/- 0.6 fmol/mg protein, n = 30) and ventricles (4.4 +/- 0.5). In contrast, methionine-enkephalyl-arginyl-phenylalanine (MEAP) immunoreactivity (IR) is higher and preferentially concentrated in the ventricle (112 +/- 12) vs. the atria (23.2 +/- 2.6 fmol/mg protein). HPLC analysis suggests the atrial/ventricular difference is partly due to altered posttranslational processing. Nearly 90% of ventricular IR is comprised of MEAP (46%) and peptide B (40%) whereas these peptides represent less than half of the atrial content. A nonneuronal localization is indicated because the peptide distribution does not correspond to the catecholamine distribution. Canine left ventricular tissue sections were processed for immunohistochemistry with the MEAP antibody. Fluorescence was distributed throughout the myocytes and concentrated in ordered lines perpendicular to the myocyte longitudinal axis corresponding to the area of the intercalated disc. This suggests opioids may be important in communication between cardiomyocytes, and possibly the presence of a unique peptide secretory mechanism utilizing the intercalated disc. The relative peptide content in cat and pig hearts was similar to the dog; however, the distribution was different. Feline cardiac ME content was distributed 2:1 in favor of the ventricles and corresponded with a preferential ventricular norepinephrine distribution. The MEAP-IR pattern gave a ventricular/atrial ratio lower (3.5:1) in cat heart vs. dog (5:1). In contrast, pig heart ME and MEAP-IR ventricular/atrial ratios were reversed for both ME (1:10) and MEAP (1:2). HPLC of pig left ventricle showed that MEAP and peptide B represented 33% and 39% of the MEAP-IR, respectively. These species variations may correlate to the differences observed in cardiac function.

Changes in brain and spinal cord water content during recurrent experimental autoimmune encephalomyelitis in female Lewis rats.

Regional changes in percent water content, a measure of regional levels of edema, were determined in female Lewis rats during key stages of recurrent experimental autoimmune encephalomyelitis (rEAE). The changes in percent water content of the spinal cord and brainstem closely paralleled the clinical and, to a lesser extent, histological course of rEAE (increasing during exacerbations and decreasing during remissions), whereas the percent water content of the forebrain, thalamus/midbrain, hypothalamus, and cerebellum remained constant and equal to control levels at all stages of the disease process. These results suggest that edema formation and resolution in the brainstem and spinal cord may be significant determinants of the transient and recurrent course of neurological dysfunction exhibited by rats with rEAE.

Surface membrane remodeling following removal of vasopressin in toad urinary bladder.

Vasopressin (ADH) increases transepithelial water flow in renal epithelia by a process that involves the insertion of water channels into the apical membrane. The objective of the present study was to examine membrane surface remodeling under conditions that promote the recovery of water channels. Hemibladders were set up as sacs with an imposed osmotic gradient. The control sacs received no hormone treatment, whereas the other sacs were stimulated with 100 mU/ml ADH for 10 or 15 min to induce exocytosis and enhanced water flow. ADH was then washed from the tissues with fresh buffer rinses to abolish the hormone actions. These tissues were then allowed to recover for 15, 30 and 60 min. During this time water channels are recovered intracellularly by a process of endocytosis. This time period was called the retrieval period. At specified time intervals, tissues were fixed and processed for SEM or embedded in epon for ultrathin sectioning for TEM studies. Control tissues, regardless of the length of time, showed little or no sign of surface remodeling that was indicative of endocytosis during pre- or post-buffer washes, whereas the ADH-treated tissues showed a time-dependent remodeling of the apical membrane during activation and following removal of the hormone during the retrieval period. At the 10 min retrieval period, greater than 47% of the granular cells showed extensive surface remodeling. By 30 and 60 min posthormone treatment during recovery, fewer than 23% of granular cells showed signs of surface membrane changes. During retrieval the apical membrane undergoes a transition with a loss of both microridges and microvilli prior to membrane restoration. These observations suggest that apical membrane remodeling is crucial for the restoration of membrane permeability following hormone activation and termination.

Modulation of cytoskeletal organization and cytosolic granule distribution by verapamil in amphibian urinary epithelia.

The present study examines the role of calcium in modulating epithelial cytomorphology by using verapamil, a calcium antagonist, and considering its effects on cytosolic granule distribution and exocytosis in toad urinary bladder. The effect of verapamil on the detection and distribution of microfilaments in toad urinary bladder using immunogold labeling techniques in toad urinary bladder epithelial cells was also examined. Verapamil, which inhibits antidiuretic hormone (ADH)-mediated water flow, increased the number, size and distribution of dense calcium-containing secretory granules in bladder epithelial cells. This calcium antagonist prevented granule exocytosis, such that, six-times the number of granules were present in verapamil-treated tissues. The normal cytomorphological changes that accompany the actions of ADH were attenuated by verapamil, including ADH-induction of microvilli. ADH increased the number of actin microfilaments as determined using protein A-gold by immunolabeling, whereas, verapamil treatment was unremarkable as compared to control. The results suggest that calcium may play a prominent role in mediating granule exocytosis and membrane fusion events that normally accompany hormone action.

In vivo degradation of oxidized, regenerated cellulose.

Oxidized, regenerated cellulose (ORC) was surgically implanted on the uterine horns of rabbits, and its biodegradation was studied in vivo. Samples of peritoneal lavages, serum, and urine were collected during the degradation process and analyzed for carbohydrate components utilizing high-performance liquid chromatography with pulsed amperometric detection (h.p.l.c.-p.a.d.). Degradation was rapid, and oligomeric products were evident primarily in the peritoneal fluid from the implantation site, with no apparent accumulation in either the serum or the urine. The size distribution and the amount of the oligomeric products decreased after day one, and by day four peritoneal lavages were essentially free of oligomers. The structure of the products formed was consistent with the lability of the polymer in solution, and the kinetics of degradation paralleled the results of the previously reported in vitro studies. Rabbit peritoneal macrophages, when incubated with ORC in vitro were observed to readily ingest and hydrolyze the polymeric material. A mechanism of degradation consisting of chemical depolymerization, followed by enzymatic hydrolysis mediated by glycosidases endogenous to peritoneal macrophages, is proposed.

Ultrastructure of a transmembrane glycoprotein, glycophorin A.

The major sialoglycoprotein of the human red cell membrane, glycophorin A, was isolated and examined by rotary shadowing and transmission electron microscopy. The glycophorin A molecule appeared as a cloud-like structure with a short, dense core within a large cloud. Mild acid hydrolysis in 0.05 M H2SO4, 80 degrees C for 1 hr reduced the size of the cloud significantly but left the dense core intact indicating that the original cloud represented the sialylated oligosaccharide chains of glycophorin A with the dense core being the polypeptide chain and its associated linkage proteins. Incubating glycophorin A with cationized ferritin (CF) revealed that the CF was bound only to the cloud, a finding that supports the view that the cloud is comprised of the sialylated oligosaccharide chains of the glycophorin A molecule. SDS-polyacrylamide gel electrophoresis revealed that our preparation of glycophorin A, as well as commercial preparations, consisted of monomers, dimers and oligomers of glycophorin A with trace amounts of the minor glycophorins and linkage proteins. Knowledge of the ultrastructure of this important integral protein will enable one to design studies to determine its functional role in the membrane.

Morphometric analysis of epithelial cells of frog urinary bladder, II. Effect of ADH, calcium ionophore (A23187) and verapamil on isolated dissociated cells.

Isolated frog urinary bladder epithelial cells, upon dissociation lose their polarity and develop microridges and occasional microvilli in a global fashion. These cells, when exposed only to isotonic Ringer's solution manifest a membrane conformation with smooth discontinuous microridges, a cytoplasm with numerous free ribosomes, rough ER, thin Golgi cisternae, mitochondria, small vacuoles, electron-dense granules, few microtubules, and numerous microfilaments and intermediate filaments with an apparent random distribution, the dissociated cells, when treated with ADH or calcium ionophore (A23187), have the appearance of numerous elongated microvilli over the entire cell surface. The cytoplasm, under these conditions, is occupied by large vacuoles with a distribution of long profiles of aggrephores and associated vesicles. The peripheral cytoplasm as well as the cavities of the elongated microvilli of these cells contain large concentrations of microfilaments often showing a strong axial orientation to the long axis of the microvilli. Many of these filamentous elements appear in contact with the apical membrane of these microvilli with an alignment with the external glycocalyx. There is an indication that these morphocytological changes as revealed by SEM and TEM studies, correlated with a redistribution and realignment of microfilaments and possibly microtubules as detected by fluorescent microscopy using immunofluorescent antibody labeling for actin and tubulin. Cells treated with verapamil, a calcium antagonist, presented dwarf and stout microvilli with little detectable alterations in the cytoplasmic compositions from that of non-hormonal treated cells. Verapamil prevented ADH induction of microvilli, with the membrane, under these conditions, appearing as compact microridges. The results indicate that calcium ionophore, like ADH, produces intense formation of microvilli in dissociated cells, mobilization and realignment of microfilaments, microtubules, increase in the density of vesicles, aggrephores and possibly secretory granules, whereas the calcium antagonist, verapamil, opposes these actions. The results suggests a prominent role of calcium in the morphological changes induced by ADH.

A comparison of wheat germ agglutinin binding between normal and sickle red blood cells.

Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.

Morphometric analysis of epithelial cells of frog urinary bladder. I. Effect of antidiuretic hormone, calcium ionophore (A23187) and PGE2.

Changes in epithelial cell morphology, especially at the apical plasma membrane, are frequently cited as initial evidence for antidiuretic hormone (ADH)-induced increase in membrane permeability. The effects of ADH and agents that alter and modify calcium and prostaglandin concentrations on the morphology and cytology of the epithelial cells of frog (Rana pipiens) urinary bladder are presented using the techniques of transmission and scanning electron microscopy. It was found that, like ADH, calcium ionophore, A23187, produce intense microvilli formation, microfilament mobilization and an increase in the density of granules and membrane associated vesicles, suggesting a prominent role of calcium in these processes. Moreover, our results suggest that these membrane and cytosolic transformations may be mediated in part through prostaglandin formation, as exogenous PGE2 mimicked these effects, and indomethacin, a prostaglandin synthesis inhibitor, attenuated ionophore's effect on luminal cytomorphology. However, unlike ADH, prostaglandins and ionophore inhibit hormonal-induced increase in transepithelial water flow. These results suggest that other components more distal to the luminal membrane, perhaps the basolateral membrane, may be rate-limiting for transepithelial water flow and possibly are regulated by either changes in calcium concentrations or prostaglandins.

Inhibition of the hydro-osmotic response to vasopressin and hypertonicity by phenothiazines and W7, calmodulin antagonists.

Phenothiazines and W7, calmodulin antagonists, inhibit the water flow response produced by ADH, exogenous cyclic AMP, phosphodiesterase inhibition and serosal hypertonicity. Calmodulin antagonists had no effect on osmotic water movement in the absence of hormone. Calmodulin was isolated and localized by immunofluorescence in bladder epithelial cells. Phenothiazines inhibited toad bladder calmodulin activation of phosphodiesterase and prevented fluorescent antibody recognition. The results suggest that the calcium-calmodulin process plays a role in the hydro-osmotic response to ADH and that produced by serosal hypertonicity. The calmodulin common site occurs subsequent to cyclic AMP formation, perhaps on the microtubule-microfilament system.