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Biomedical subjects

C G Wigham

Publications and source records attributed to C G Wigham.

12 recordsLinked to original sources
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↗
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↗
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

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↗
PubMed1993

Rabbit corneal endothelial cell membrane potential.

Membrane potential of rabbit corneal endothelial cells measured using microelectrodes was -29.3 +/- 0.8 mV, n = 45, (mean +/- SEM). Histological location of Lucifer Yellow dye iontophoresed out of the microelectrode confirmed that the microelectrode was located intracellularly. The Lucifer Yellow diffused five to six cell diameters away from the impaled cell indicating endothelial cell coupling. Depolarization by ouabain (10(-4) M) and high extracellular potassium (potassium for sodium substitution) showed the cells to be responsive to changes in the bathing solution whilst impaled, that the cell membrane is more permeable to potassium than sodium and that membrane bound Na(+)-K(+)-ATPase activity generates the transmembrane electrolyte gradients.

Animals↗
PubMed1987

Physiological changes in the cornea of the ageing eye.

Donor human corneas were studied in vitro for the viability of their endothelia. Endothelial ionic permeability (measured by electrical conductivity) and bicarbonate pump activity (measured by short circuit current) were recorded. Paired corneas were used to monitor post-mortem deterioration times and drug therapy immediately prior to death noted, when possible, to identify any possible effect on tissue performance. Donor age was correlated with some loss of endothelial pump capability. In adults it seems that pump capability deteriorates with a half time decay of 72 years (correlation coefficient, 0.69). Typically, between 60 and 90 years, endothelial pump capability may drop from 32 mu amps.cm-2 to 22 mu amps.cm-2, although at any particular age, the natural variation is +/- 6 mu amps.cm-2. Over the same period, endothelial permeability does not deteriorate and there is a suggestion that the endothelial cells may even tighten up with ageing, thereby partially compensating for the suggested ageing diminution in metabolic pump capability. There is no obvious age threshold for endothelial deterioration. The use of paired corneas revealed a 50 per cent loss in endothelial barrier function per 4.8 days of storage, and less than 5 per cent loss of pump capability over a similar period. The eyes were stored whole in a moist chamber at 0-4 degrees C. A significant group of donor eyes had a markedly reduced pump capability which was not age related. The patients had had terminal drug treatments which included digoxin and furosemide.

Aged↗
PubMed1987

Paracellular ionic and transcellular water diffusions across rabbit corneal endothelium.

1. Corneal endothelial cell membrane electrical resistance is estimated at about 400 M omega by using intracellular micro-electrodes. 2. If all ion diffusion across the endothelium were transcellular, electrical resistance of the endothelial monolayer would be about 1300 omega cm2, as there are about 3000 cells mm-2. 3. Measured endothelial monolayer resistance is 12.7 +/- 0.8 omega cm2 (mean +/- S.E., n = 6), which indicates that about 99% of ion diffusion does not cross the transcellular pathway, but must pass through the paracellular route. Arguments are presented which suggest that the proportion may be even higher. 4. Endothelial passive ion permeabilities are about the same as corneal stromal passive ion permeabilities. 5. Stromal water diffusional permeability is about the same as stromal ion permeability, after allowance is made for free diffusion coefficients. In contrast, endothelial water diffusional permeability is so high as to be unmeasurable. 6. It is concluded that ions diffuse across the corneal endothelium through the paracellular route, and that water diffuses across the endothelium mainly through the transcellular route.

Animals↗
PubMed1984

The promotion of mitosis in cultured thymic lymphocytes by acetylcholine and catecholamines.

Thymic lymphoblasts possess beta-adrenergic, dopaminergic and nicotinic receptors. When activated by high concentrations of adrenaline, isoprenaline, dopamine and acetylcholine, magnesium-dependent events are initiated, which culminate in mitosis. These events can be blocked by testosterone. The cells also possess muscarinic and alpha-adrenergic receptors which respond to low concentrations of acetylcholine, carbamylcholine and noradrenaline. In these cases calcium-dependent, oestradiol-blockable mechanisms are triggered which eventually lead to cell division.

Acetylcholine↗