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Biomedical subjects

S A Hodson

Publications and source records attributed to S A Hodson.

At least 19 recordsLinked to original sources
PubMed2004

Rabbit corneal hydration and the bicarbonate pump.

Experiments were conducted on the transport properties of the rabbit corneal endothelium at 22 degrees C, at which temperature the endothelium was able to stabilize the hydration of corneal stroma at physiological values. When bicarbonate was omitted from the bathing solution, the cornea swelled at 11 +/- 1 microm x h(-1). The swelling was completely reversible upon the subsequent re-introduction of bicarbonate. Similar swelling rates were observed when the endothelial pump was irreversibly inhibited with ouabain. In an Ussing-type chamber, the endothelium developed an electrical resistance of 25.0 +/- 1.0 ohms x cm2 and a short circuit current (s.c.c.) of 6.0 +/- 1.1 microA x cm(-2). Neither electrical resistance of the corneal endothelium nor its s.c.c. were changed significantly after exposure to 0.5 mM amiloride. Ouabain abolished the s.c.c. but had no significant effect on resistance. When paired preparations were short-circuited, the endothelium developed a net H[14C]O3- flux of 0.24 +/- 0.03 micromoles x cm(-2) x h(-1) into the aqueous humour, which was close in magnitude and direction to the s.c.c. of 0.22 +/- 0.01 microEq x cm(-2) x h(-1). There was no significant net flux of 86Rb (0.04 +/- 0.03 micromoles x cm(-2) x h(-1)). Similar magnitude fluxes for both bicarbonate and rubidium were found with open-circuit preparations. It is suggested that a metabolically driven electrogenic bicarbonate current passing across the corneal endothelium is solely responsible for maintaining corneal hydration at 22 degrees C. Based on these and other studies, a model is proposed for active bicarbonate transport across corneal endothelium consisting of uphill entry into the cell through a baso-lateral membrane sodium/bicarbonate cotransporter (NBC) and downhill exit through an apical membrane anion channel. Studies on the transport properties of the endothelium at 35 degrees C are discussed and reasons suggested for the discrepancy between short circuit current and net bicarbonate flux at this closed eye temperature.

Animals↗
PubMed2004

Structural changes in alpha-crystallin and whole eye lens during heating, observed by low-angle X-ray diffraction.

Whole eye lens and alpha-crystallin gels and solutions were investigated using X-ray scattering techniques at temperatures ranging from 20 degrees C to 70 degrees C. In whole lens isolated in phosphate-buffered saline, the spacing of the dominant X-ray reflection seen with low-angle scattering was constant from 20 degrees C to 45 degrees C but increased at 50 degrees C from 15.2 nm to 16.5 nm. At room temperature, the small-angle X-ray diffraction pattern of the intact lens was very similar to the pattern of alpha-crystallin gels at near-physiological concentration (approximately 300 mg/ml), so it is reasonable to assume that the alpha-crystallin pattern dominates the pattern of the intact lens. Our results therefore indicate that in whole lens alpha-crystallin is capable of maintaining its structural properties over a wide range of temperature. This property would be useful in providing protection for other lens proteins super-aggregating. In the alpha-crystallin gels, a moderate increase in both the spacing and intensity of the reflection was observed from 20 degrees C to 45 degrees C, followed by an accelerated increase from 45 degrees C to 70 degrees C. Upon cooling, this effect was found to be irreversible over 11 hours. Qualitatively similar results were observed for alpha-crystallin solutions at a variety of lower concentrations.

Animals↗
PubMed2004

The ordering of corneal collagen fibrils with increasing ionic strength.

The fixed stromal charge of bovine corneas, osmotically clamped at physiological hydration, was altered by regulating the amount of chloride ions bound to the matrix. We measured the local fibrillar collagen order using X-ray diffraction methods. As the bound anions increased up to physiological values, the local fibrillar order increased to an optimal value. The coherence distance (t) approximately doubles to a maximum value (409 nm) from 10 mM NaCl to 154 mM NaCl. This then slowly decreased as the bathing solution increased to 1000 mM. In contrast the diameter of the collagen fibrils were minimal at physiological NaCl.

Animals↗
PubMed2003

Neutron and X-ray scattering by ox corneal stroma differentially loaded with bound anions.

Ox corneas at near physiological hydration were subjected to two variables: the amount of chloride ions bound to them and exposure of various mixtures of H(2)O/D(2)O as solvent. The preparations were then exposed to a neutron beam and the contrast match points, at which the collagen fibrils of the corneal stroma most nearly matched the scattering density of the various H(2)O/D(2)O mixtures, were measured. In both cases of high and low bound chloride, the contrast match points of the collagen fibril were equal, indicating that there were no significant changes in the water of electrostriction at the fibril surface when chloride ions bind to the stroma. The data suggest that the ligands which bind anions to corneal stroma are not located at the collagen fibril surface. When the chloride binding ligands were extracted from the corneal stroma there were significant changes in the structure of the fibrils. We suggest that the chloride binding ligands may be located within the collagen fibril.

Animals↗
PubMed2000

Modulation of corneal endothelial hydration control mechanisms by Rolipram.

Corneal stromal hydration is maintained by an active HCO3- transport mechanism located in the corneal endothelium. Whilst modulation of transport activity by changes in intracellular cAMP concentration have been noted, the site of effect is undefined. To resolve this question, the effects of Rolipram, a cAMP phosphodiesterase inhibitor, on endothelial physiology were determined. Addition of 0.1 mM Rolipram caused a threefold increase in intracellular cAMP with no change in cGMP. Associated with the increase in cAMP was a transient whole corneal thinning and a similarly transient increase in trans-endothelial potential difference, short-circuit current and resistance. The membrane potential hyperpolarized and the intracellular Na+ concentration decreased. The decreased intracellular Na+ was associated with an increased rate of Na+ extrusion between the endothelial cell and extracellular space. It is concluded that Rolipram increases the concentration of cAMP which activates the basolateral membrane Na+/K+-ATPase activity and increases net HCO3- transport. In addition there is a reduction in endothelial permeability which combined with the increase in pump activity may jointly explain the observed stromal thinning. The duplicity of responses indicates that if cAMP has a physiological role in regulating corneal hydration then it may operate on both the endothelial pump and the endothelial permeability.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

A chloride-activated Na(+)/HCO(3)(-)-coupled transport activity in corneal endothelial membranes.

Investigations of corneal endothelium were made to resolve the apparent contradiction of the presence of sodium/bicarbonate cotransporter (NBC) in fresh and cultured cells and NBC's reported absence in isolated plasma membrane vesicles. Gradient-driven ion fluxes into the vesicles were measured. Short-term incubations (0-30 s) showed the presence of a bicarbonate-dependent inward sodium flux (BDSF), which was active when the insides of the vesicles were preloaded with chloride ions. The BDSF was absent if chloride was present only externally to the vesicles. Chloride at concentrations between 30 and 40 mM inside the vesicle had its maximum effect on BDSF. Other anions (acetate, thiocyanate, or gluconate) inside the vesicles did not mimic the chloride effect. Associated with the net inward sodium flux was a net inward bicarbonate flux. Hill plots of sodium influx with respect to external bicarbonate concentrations indicated that the stoichiometry of the net transfer was 1.7 +/- 0.2 (mean +/- standard error, n = 5) bicarbonate ions for each sodium ion transported. There was no net chloride flux found across the membrane vesicles. The finding of a novel chloride-activated NBC activity fully resolves the apparent contradiction between whole-cell and membrane vesicle preparations.

Animals↗
PubMed1999

Sodium ion uptake into isolated plasma membrane vesicles: indirect effects of other ions.

Vesicles derived from plasma membrane of corneal endothelium were agitated to their minimum size distribution. When isotonic salt solutions surrounding the vesicles were changed there were alterations to the vesicle size distribution: the modal point of the logarithmic distribution did not change but the log variance did, indicating that substantial fission and fusion of vesicles occurred depending upon the nature of the surrounding solute. Orientation and total membrane area was conserved in the transformed population of vesicles. Although the ions added to the external isotonic salt solutions in the present series of experiments have no direct effect upon sodium membrane transporters in these membranes, kinetics of sodium accumulation into the vesicles were affected in a way that correlated with changes to the vesicle size distribution. Early-saturating (<1 min) intravesicular concentrations of sodium corresponded with apparently stable populations. Late-saturating (>1 min) intravesicular concentrations of sodium corresponded with significant vesicle distribution shifts and included a few seconds of delay. During the linear accumulation phase, both populations showed similar magnitudes of sodium transport. The significance of these data is discussed.

Animals↗
PubMed1999

A rapid method of preparing the plasma membrane enriched fraction of the bovine retinal pigment epithelial cells.

PURPOSE: To devise a rapid method of isolating the plasma membrane enriched fraction (PMEF) of the bovine retinal pigment epithelial (RPE) cells with Percoll centrifugation medium. METHODS: The bovine RPE was homogenised with a tight fit Dounce homogeniser and centrifuged in a 16.7% Percoll gradient for 20 minutes. The RPE particulate fractions were characterised in terms of their protein concentrations, Na/K-ATPase and bicarbonate stimulated ATPase activities. RESULTS: The total protein recovery was 88.7% of the RPE homogenate. The nucleus layer was identified at the first band. The mitochondrial fraction was at the second layer according to its bicarbonate stimulated ATPase activity. The 3rd and 4th bands were enriched with plasma membranes and their Na/K-ATPase activities were 31.5 and 34.6 mumol/mg/h respectively. The Na/K-ATPase activities were about six times that of the RPE homogenate. CONCLUSIONS: A rapid method of isolating the bovine RPE PMEF has been devised which involved a single centrifugation procedure in a Percoll gradient.

Animals↗
PubMed1998

The glucose transport in retinal pigment epithelium is via passive facilitated diffusion.

The glucose transport across the bovine retinal pigment epithelium (RPE) was studied in a modified Ussing chamber. Unidirectional fluxes were recorded with radioactive tracers L-[14C]-glucose (LG) and 3-O-methyl-D-[3H]-glucose (MDG). There was no significant difference between the unidirectional MDG fluxes (retina to choroid, and choroid to retina directions) with or without ouabain. The effects of two glucose transporter inhibitors, phloretin and cytochalasin B, on the glucose fluxes from choroid to retina cells were also investigated. The MDG flux was found to be inhibited by 45.5% by phloretin (10(-4) M) and 87.4% by cytochalasin B (10(-4) M). These inhibitory characteristics resembled the facilitated diffusion mode of glucose transport. The glucose transporter protein in the plasma membrane of RPE was located by means of photolabeling [3H]-cytochalasin B. The labeled plasma membrane enriched fraction was analysed by SDS-PAGE. The glucose transporter of bovine RPE was found to have a molecular weight range of 46-53 kDa. The molecular weight range of this transporter protein agreed with those of facilitated glucose transporters in other tissues indicating a molecular similarity between them. The results indicated that the glucose transport across the RPE is via passive facilitated diffusion.

Animals↗
PubMed1997

Determination of Na+/Cl-, Na+/HCO3- and Na+/K+/2Cl- co-transporter activity in corneal endothelial cell plasma membrane vesicles.

Corneal endothelial cell derived plasma membrane vesicles were used to investigate the presence of Na+/Cl-, Na+/HCO3- and Na+/K+/2Cl- co-transporter activity in the plasma membranes of these cells. Na+/H+ exchange was blocked by the presence of 1 mM amiloride in all determinations. The rate of accumulation of Na+ in the presence of chloride or bicarbonate was not significantly different from its accumulation in the presence of acetate, thiocyanate or gluconate. The addition of K+ to Na+ plus Cl- did not stimulate Na+ accumulation into the vesicles. The present work provides no evidence for Na+/K+/2Cl-, Na+/Cl- or Na+/HCO3- co-transport in corneal endothelial cell plasma membrane vesicles.

Animals↗
PubMed1996

Two pathways for electrogenic bicarbonate ion movement across the rabbit corneal endothelium.

Amiloride (0.5 mM) inhibited the rate of entry of Na+ into corneal endothelial cells by more than half ((0.76 +/- 0.10) to (0.21 +/- 0.10) microEq cm(-2)h(-1)). The same concentration of amiloride caused only minimal disturbance to corneal hydration control by the endothelium (range 0-12%). Amiloride (0.5 mM) and acetazolamide (1 mM) reversibly inhibited trans-endothelial short circuit current by about a half. Their combined effect was not additive. Acetazolamide (1 mM) reduced net HCO3- flux across the short-circuited endothelium by about the same amount ((0.50 +/- 0.11) microEq cm(-2)h(-1)) that amiloride (0.5 mM) reduced Na+ entry into the cells ((0.55 +/- 0.14) microEq cm(-2)h(-1)). Low concentrations of amiloride (10 microM) had little effect on the transport characteristics of the endothelium, indicating that Na+ entry into the endothelial cells under physiological conditions is not primarily through Na+ channels. The data are consistent with an Na+/H+ exchanger acting in tandem with carbonic anhydrase through a pathway which could have a regulatory role on endothelial transport via its effect on Na+ re-entry. Residual trans-endothelial HCO3- transport, apparently unaffected by amiloride or acetazolamide inhibition, is calculated to be of sufficient magnitude to maintain corneal hydration.

Acetazolamide↗
PubMed1995

Chloride binding in the stroma of cultured human corneas.

In the ox cornea, more than half of the non-diffusible, matrix negative charge is derived from the binding of free chloride ions. Because the magnitude of the net matrix charge is the dominant factor which determines the degree of stromal swelling, we investigated whether this phenomenon, stromal chloride binding, also occurs in human corneal stroma. Intrastromal ion concentrations were measured with radio-isotopes when human (outdated Eye Bank) corneas or (fresh) bovine corneas, physically clamped to maintain a constant hydration, were incubated in buffered 154 mM NaCl. The intrastromal chloride ion concentration was compared to the normalized concentrations of trace quantities of radio-labelled acetate and lactate ions. For human corneas, the intrastromal chloride ion concentration was found to be significantly higher (P < 0.001, t-test) than the normalized concentrations of both acetate and lactate ([Cl]i = 142.5 +/- 0.9 mM, (n = 9); [acetate]i = 131.2 +/- 1.2 mM, (n = 8); [lactate]i = 131.9 +/- 1.5 mM, (n = 5); all values are mean +/- S.E.M.). The sodium ion concentration was elevated ([Na]i = 176.0 +/- 1.8 mM, (n = 9)). These results demonstrate that chloride binding occurs to a significant extent in cultured human corneal stroma and suggest that chloride binding may be evident in the native human cornea.

Animals↗
PubMed1994

Determination of pathways for sodium movement across corneal endothelial cell derived plasma membrane vesicles.

A bovine corneal endothelial cell plasma membrane vesicle preparation was used to investigate passive Na+ transport across the plasma membrane of these cells. Sodium accumulation rate into the vesicle was not dependent on the presence of HCO3- or a HCO3- gradient, but was stimulated by a trans-vesicle pH gradient. Amiloride, furosemide and DIDS all reduced the rate of Na+ accumulation. The data indicate the presence of at least two independent pathways for passive sodium movement across the vesicle: the first probably via a Na+/H+ exchanger and the second a furosemide inhibitable Na+ entry mechanism. No evidence was found for direct Na(+)-HCO3- coupled transport.

Amiloride↗
PubMed1994

Localization of Na+/K(+)-ATPase in the bovine corneal endothelium.

A mouse monoclonal antibody has been used to localize Na+/K(+)-ATPase in the bovine corneal endothelium. The specificity of the antibody was demonstrated by reaction with a single protein of molecular mass 100 kDa on Western blots and immunoprecipitation of a complex consisting of 100 kDa and 50 kDa subunits. Treatment of the immunoprecipitated antigen with Peptide N-Glycanase F produced no change in the molecular mass of the 100 kDa protein, but resulted in a progressive decrease in the molecular mass of the 50 kDa subunit, to yield a core protein of molecular mass about 33 kDa. The pattern of deglycosylation suggested the presence of three N-linked glycans attached to the 33 kDa protein core. These results were consistent with the antibody being specific for the alpha subunit of the Na+/K(+)-ATPase. Immunocytochemical studies at the light and electron microscopic level demonstrated antibody binding to both the basal and lateral membranes of bovine corneal endothelial cells. This suggested a baso-lateral distribution of Na+/K(+)-ATPase in these cells, rather than the previously proposed lateral membrane-only distribution.

Animals↗
PubMed1994

Sodium movement into and out of corneal endothelium.

Rabbit corneal endothelial cells mounted in vitro were impaled simultaneously with Na(+)-selective and conventional KCl-filled microelectrodes. The membrane potential (Vm) was -30.4 +/- 0.8 mV (mean +/- SEM, n = 55) and the intracellular [Na+]i (calculated from the Na(+)-selective electrode potential, VNa) was 13.7 +/- 1.9 mM (mean +/- SEM, n = 16). When ouabain was added to the perfusate the cell depolarised, causing both Vm and VNa to increase with a very similar time course. Final Vm was -6.3 +/- 0.6 mV (mean +/- SEM, n = 15), and the final [Na+]i was 114 +/- 6.9 mM (mean +/- SEM, n = 5). The parallel increase in Vm and rise in [Na+]i suggest that a component of the ouabain-induced depolarisation of the cell (increase in Vm) is due to Na+ entry into the cell down its concentration gradient. The lateral and basal location of the Na+/K(+)-ATPase in bovine endothelial cells was confirmed (for the first time at the electron-microscopic level) using a monoclonal antibody specific for the alpha 1 subunit of Na+/K(+)-ATPase. The absence of a net Na+ flux across these cells combined with the basolateral location of the ATPase suggest that Na+ exit from the cell, and its re-entry take place across the same membrane (i. e. the basolateral).

Animals↗
PubMed1991

Evidence for keratin proteins in normal and abnormal human meibomian fluids.

Hyperkeratinization of meibomian glands has been postulated to cause gland dysfunction. Recent investigations on rabbits show that keratin proteins are indeed present in the meibomian fluids of these animals. In this report we present our findings on the presence of these water-insoluble proteins in human meibomian secretions. 6 anti-cytokeratin antibodies, CK8, 18, 19, CK7, CK8, CK14, CK19 and AE1/AE3 were used against the keratin proteins expressed from the human meibomian fluids. Using the immunoblotting (dot blot) technique, abnormal waxy meibomian fluids obtained from subjects diagnosed to have meibomian gland dysfunction (MGD) were compared to normal clear meibomian fluids. The results show that keratins are present in a higher concentration (10%) in the abnormal human meibomian excreta as compared to the normals. Even though the presence of protein markers for keratinization in the abnormal meibomian excreta were not shown, the increased presence of keratin proteins in the abnormal meibomian fluids suggests that, in MGD patients, hyperkeratinization of ductal epithelium may have taken place. More keratin proteins (possibly those of higher molecular weights) were produced in addition to the keratin proteins normally produced by the duct epithelium. The increased amount of keratin proteins in the abnormal meibomian fluids may be explained by the susceptibility of duct epithelium to undergo the process of hyperkeratinization as postulated by other researchers.

Antibodies, Monoclonal↗