David R. Whikehart

Tritiated thymidine experiments in the cat: a description of techniques and experiments to define the time-course of radioactive thymidine availability

Tritiated thymidine [(3H]thymidine) autoradiographic techniques have been used to define birthdates for cells in a variety of animals. In an effort to include [3H]thymidine experiments in our ongoing studies of visual system development, we have used an intrauterine injection procedure that affords [3H]thymidine labeling of dividing nerve cells in the cat. This report contains a detailed description of the injection procedures used, as well as the results of experiments undertaken to define the period of time during which the exogenous [3H]thymidine introduced by such injections remains available for uptake.

The effects of glutathione and adenosine on plasma membrane ATPases of the corneal endothelium. An hypothesis on the stimulatory mechanism of perfused glutathione upon deturgescence

Reduced glutathione (0.3 mM) stimulates the activity of sodium-potassium activated ATPase (Na+K+ATPase) by 54% in plasma membranes prepared from bovine corneal endothelial cells. Oxidized glutathione, however, has no effect on Na+K+ATPase activity in the same tissue, although it does inhibit magnesium activated ATPase (Mg++ATPase) by approximately 30%. Adenosine neither stimulates nor inhibits either Na+K+ATPase or Mg++ATPase in these plasma membranes. It is postulated that the stimulatory effect of glutathione on deturgescence stems from the direct reaction of the reduced form of the tripeptide on sulfhydryl groups located on plasma membranes of corneal endothelial cells. It is highly probable that these sulfhydryl groups are part of the Na+k+ATPase complex itself.

Angiogenic activity of an Onchocerca volvulus Ancylostoma secreted protein homologue

Angiogenesis is an important step in the development of ocular onchocercaisis. In previous studies, it has been demonstrated that Onchocerca volvulus homologues of the Ancylostoma secreted protein family have pronounced angiogenic activity. The overall goal of the current study was to determine if this angiogenic effect is exerted through a direct or indirect mechanism. These studies focused on one member of this family, OvASP-2, as this protein is expressed in microfilaria, the stage of the parasite that causes ocular onchocercaisis. Clones encoding truncated and full length open reading frames were expressed as fusion proteins with Escherichia coli maltose binding protein (MBP), and angiogenic activity was compared in vitro and in vivo with MBP alone. Truncated constructs expressing only the first 105 amino acids of OvASP-2 were as active as the full length protein in inducing new blood vessel formation. The full length fusion protein did not stimulate proliferation or production of vascular endothelial growth factor in vascular endothelial cells in vitro, indicating that OvASP-2 does not directly stimulate angiogenesis. Sequence analysis demonstrated that the gene encoding OvASP-2 contained five introns. Sequence comparisons of the genomic loci from West African blinding and non-blinding strains of O. volvulus revealed that some polymorphism existed among the various isolates tested. However, none of these polymorphisms could be used to differentiate the parasite strains, suggesting that qualitative variation in OvASP-2 could not explain the difference in ocular pathogenic potential of the two parasite strains.

Effects of mannitol on cultured corneal endothelial cell Na,K-ATPase activity.

Mannitol was introduced into the media of bovine corneal endothelial cells grown in culture. This was accomplished in order to compare its influence on Na,K-ATPase activity with that of high levels of glucose that inhibit the enzyme. The study was conducted with the intent of showing possible adverse osmolar effects on enzyme activity. Mannitol was found to inhibit NA,K-ATPase when compared with mannitol-free medium (11 U vs. 202 U). However, this inhibition was significantly greater than that produced by high levels of glucose. When mannitol was introduced directly into the assay for the plasma membrane- extracted enzyme, the inhibition was as severe (14 U) as for that placed in the culture medium. By way of comparison, glucose introduced into the enzyme assay caused no significant inhibition (189 U). The mannitol concentration used was 20 mM and in the media there was an osmotic pressure of 347 mOsmol/kg. The high glucose concentration was 25 mM and the osmotic pressure in the media was 341 mOsmol/kg. These osmotic pressures were compared with that of the control medium (with 5 mM glucose), which generated a pressure of 308 mOsmol/kg. None of these values were judged sufficiently high to rupture cell plasma membranes or alter cell morphology as seen by vital staining and phase contrast microscopy. In addition, it was found that mannitol had no effect on cellular DNA, whereas a previous study showed that high glucose caused an increased unwinding of duplex DNA. This study suggests that mannitol inhibits endothelial cell Na,K-ATPase by a different mechanism than that of glucose. It further points out that osmotic effects may not be involved with either mechanism.

Sodium and potassium saturation kinetics of Na+K+-ATPase in plasma membranes from corneal endothelium: Fresh tissue vs. tissue culture

The maximal velocity (Vmax) and the apparent dissociation constant (K0.5) of Na+K+-ATPase have each been estimated with respect to sodium and potassium ion activation. These estimations were made from the enzymatic activity of plasma membrane preparations derived from bovine corneal endothelial cells. The determinations were made on cells obtained from fresh tissue and from secondary tissue cultures. Two methods were used to obtain the estimates: the first used a combination of Eadie-Hofstee and Hill plots; the second used Eisenthal-Cornish-Bowden plots. The Vmax for sodium was 5.58-5.60 mumol Pi/mg protein/30 min for fresh tissue vs. 2.00-1.80 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 62-57 mM (fresh tissue) vs. 7.9-6.7 mM (tissue culture). Vmax for potassium was 4.28-4.00 mumoles Pi/mg protein/30 min for fresh tissue vs. 1.37-1.34 mumoles Pi/mg protein/30 min for tissue cultures. The corresponding K0.5 values were 3.3-3.1 mM (fresh tissue) and 1.7-1.7 mM (tissue culture). The results indicate a lowered activity and change in affinity of the enzyme for the two ions in tissue cultures compared to fresh tissues. The detergent Lubrol W-X increased activity in both tissue sources. Sonication had no significant effect on the activity. Variations in pH (7-9) indicated that the highest activity was obtained at pH 7.8 for the enzyme in tissue culture while activity was highest at pH 8.0 for the enzyme in fresh tissues. These differences in kinetic activity suggest a response to changes in the ion requirements of these cells due to their environment, developmental stage or some other parameter.

Common cryopreservation media deplete corneal endothelial cell plasma membrane Na+,K+ ATPase activity

This study describes the effects of three cryopreservation media on the specific activity of corneal endothelial plasma membrane Na+,K+ ATPase activity, a transporter required for the fluid pump in the cornea. Bovine corneal endothelial cell cultures were used as a model system for these studies. Cryopreserved primary cells were thawed and passaged once to increase cell number. The specific activity plasma membrane Na+,K+ ATPase activity was subsequently measured on 4-6 replicate cultures. One freeze/thaw cycle depleted the Na+,K+ ATPase specific activity of corneal endothelial cell cultures by approximately 90%, as compared to cells of equivalent passage which had not been cryopreserved. Cell morphology of the cryopreserved cultures was indistinguishable from that of control cultures. In other experiments, first passage cultures which had not been subjected to cryopreservation were incubated with a dimethyl sulfoxide-, glycerol-, or propane diol-based freezing medium and Na+,K+ ATPase was measured on plasma membranes subsequently isolated from the cultures. Incubation of cells with cryopreservation media in the absence of the freezing process also depleted Na+,K+ ATPase by approximately 90%. Radiolabeled ouabain was used to measure Na+,K+ ATPase sites on cell cultures pretreated with dimethyl sulfoxide-based freezing media. A 4 h treatment with DMSO-based freezing medium had no effect on ouabain binding; treatment for 18 h reduced binding by only 50%. Thus, the method used to assess pump function (determination of Na+,K+ ATPase specific activity versus ouabain binding) may provide conflicting data concerning the level of pump function cultured cells. The cryoprotectants present in many common media used to freeze tissue culture cells appear to inhibit corneal endothelial Na+,K+ ATPase. Since the fluid pump of corneal endothelial cells is coupled to Na+,K+ ATPase activity, care must be taken to insure that pump function is not impaired during cryopreservation of cell cultures.

Low-molecular-weight peptides in corneal tissue culture media and in bovine aqueous fluid

Eight putative, low-molecular-weight peptides have been found in the media of bovine corneal tissue cocultures and in bovine aqueous fluid. By investigating amine fluid extracts from these sources by HPLC, possible amino acids, vitamins and urea were eliminated as the unknown peaks of interest. The molecular weights of these substances were equal to or less than 1,000 D. The peaks did not correspond to glutathione or bradykinin either which fall in this molecular weight range. As the cultures aged from 10 to 32 days, it was found that 5 of the 8 peptides varied in concentration. Five peptides increased in concentration to day 17 and slowly decreased afterwards. Efforts are not directed toward the identification of these peptides.