Four-point fixation technique for sutured posterior chamber intraocular lenses.
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Cataract Extraction↗
Biomedical subjects
B A Hughes
Publications and source records attributed to B A Hughes.
Anterior segment trauma and air bags.
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Accidents, Traffic↗
Effects of Ba2+ and Cs+ on apical membrane K+ conductance in toad retinal pigment epithelium.
Intracellular microelectrode techniques were employed to characterize the blocker sensitivity of the K+ conductance (gK) at the apical membrane of the toad retinal pigment epithelium (RPE). Increasing the K+ concentration in the apical bath ([K+]o) from 2 to 5 mM produced a rapid depolarization of the apical membrane potential (VA). The addition of 0.5 mM Ba2+ or 5 mM Cs+ to the apical bath rapidly depolarized VA and increased the transepithelial resistance and ratio of apical-to-basolateral membrane resistance. In the presence of apical Ba2+ or Cs+, the response of VA to delta [K+]o was markedly reduced, indicating that these ions are effective blockers of apical gK. The Ba(2+)- and Cs(+)-induced decreases in the apparent apical-to-basolateral membrane conductance ratio were concentration dependent, with apparent dissociation constants of 17 microM and 0.5 mM, respectively. The apparent blocker sensitivity of apical gK is similar to that previously demonstrated for the inwardly rectifying K+ conductance in isolated toad RPE cells, suggesting that the inwardly rectifying K+ conductance comprises much of apical gK.
Animals↗
An outwardly rectifying K+ current active near resting potential in human retinal pigment epithelial cells.
Currents in freshly dissociated adult human retinal pigment epithelial (RPE) cells were studied using the perforated patch-clamp technique. The zero-current potential (V0) averaged -48.9 +/- 7.7 mV (n = 50). Depolarizing voltage pulses from -70 mV evoked an outward current that activated with first-order kinetics and that did not inactivate during prolonged depolarizations. Repolarizing the membrane potential produced tail currents that reversed near the K+ equilibrium potential, indicating that the sustained outward current was carried mainly by K+. The outwardly rectifying K+ conductance (gK) had an activation threshold voltage near -60 mV and was half-maximal at -37 mV. Approximately 25% of gK was active at the average V0. The K+ current was nearly completely blocked by 2 mM Ba2+ but was relatively insensitive to 20 mM tetraethylammonium. The kinetics, voltage dependence, and blocker sensitivity of this current clearly distinguish it from delayed rectifier K+ currents previously identified in RPE cells. We conclude that the sustained outward K+ current may help establish the resting potential of the apical and/or basolateral membranes and may also participate in K+ transport across the RPE.
Adult↗
Properties of the inwardly rectifying K+ conductance in the toad retinal pigment epithelium.
An inwardly rectifying K+ current was analysed in isolated toad retinal pigment epithelial (RPE) cells using the perforated-patch clamp technique. The zero-current potential (Vo) of RPE cells averaged -71 mV when the extracellular K+ concentration ([K+]o) was 2 mM. Increasing [K+]o from 0.5 to 5 mM shifted V0 by +43 mV, indicating a relative K+ conductance (TK) of 0.74. At [K+]o greater than 5 mM, TK decreased to 0.53. Currents were larger in response to hyperpolarizing voltage pulses than depolarizing pulses, indicating an inwardly rectifying conductance. Currents were time independent except in response to voltage pulses to potentials positive to 0 mV, where the outward current decayed with an exponential time course. Both the inwardly rectifying current and the transient outward current were eliminated by the addition of 0.5 mM Ba2+, 5 mM Cs+ or 2 mM Rb+ to the extracellular solution. The current blocked by these ions reversed near the K+ equilibrium potential (EK) over a wide range of [K+]o, indicating a highly selective K+ channel. The current-voltage relationship of the isolated K+ current exhibited mild inward rectification at voltages negative to -20 mV and a negative slope conductance at voltages positive to -20 mV. The Cs(+)- and Ba(2+)-induced blocks of the K+ current were concentration dependent but voltage independent. The apparent dissociation constants were 0.8 mM for Cs+ and 40 microM for Ba2+. The K+ conductance decreased when extracellular Na+ was removed. Increasing [K+]o decreased the K+ chord conductance (gK) at negative membrane potentials. In the physiological voltage range, increasing [K+]o from 2 to 5 mM caused gK to decrease by approximately 25%. We conclude that the inwardly rectifying K+ conductance represents the resting K+ conductance of the toad RPE apical membrane. The unusual properties of this conductance may enhance the ability of the RPE to buffer [K+]o changes that take place in the subretinal space at the transition between dark and light.
Animals↗
Exogenous endophthalmitis initially treated without systemic antibiotics.
PURPOSE: In 1987, the authors reported the successful eradication of infection in 16 patients with culture-proven acute exogenous bacterial endophthalmitis using intravitreal but no systemic antibiotics. They retrospectively reviewed additional consecutive cases since then to determine if the initial omission of systemic antibiotics remained reasonable. METHODS: Twenty patients had culture-proven endophthalmitis. Four patients initially received systemic antibiotics for orbital cellulitis (2 patients), prevention of a possible scleral buckle infection (1 patient), and ascending cholangitis (1 patient). The remaining 16 patients were treated initially with intravitreal antibiotics only. FINDINGS: Three of these additional 16 patients ultimately required systemic antibiotics for orbital cellulitis (1 patient), infectious scleritis (1 patient), and prevention of central nervous system infection with Neisseria meningitidis (1 patient). Only in one patient who had a neglected endophthalmitis and in whom an orbital cellulitis ultimately developed were we unable to clear the intraocular infection. In the overall series of 32 patients, cultures yielded staphylococcal species in 16 eyes, gram-positive bacilli in 3, streptococcal infection in 5, gram-negative cocci in 1, and gram-negative bacilli in 7. Half of the 14 specimens (1 aqueous and 13 vitreal) collected at the time of 16 reinjections in 13 eyes yielded organisms. Half (16/32) of the eyes attained visual acuity of 20/40 or better; 87.5% (28/32) attained visual acuity of 20/400 or better. CONCLUSIONS: Therapy with intravitreal antibiotics without systemic antibiotics is reasonable, unless the infection has extended (or is at risk to extend) beyond the globe. Such evidence includes an elevated temperature or leukocyte count, corneal ring abscess, proptosis, loss of extraocular movements, scleral abscesses or infectious scleritis, and, perhaps, the presence of a scleral buckle.
Anti-Bacterial Agents↗
Direct evidence for a basolateral membrane Cl- conductance in toad retinal pigment epithelium.
There is now evidence that a Cl- conductance on the basal membrane of the retinal pigment epithelium (RPE) is involved in the generation of both the fast oscillation and the light peak of the direct-current electroretinogram as well as being critical for transepithelial fluid and salt movement. In the present study, we characterized the basolateral membrane Cl- conductance of an in vitro preparation of toad RPE-choroid using conventional and Cl(-)-selective microelectrodes. Under control conditions, the potential across the apical (Vap) and basal (Vba) membranes averaged -60 +/- 2 and -45 +/- 2 mV, respectively (n = 40). Intracellular Cl- activity (aiCl = 20 +/- 1 mM) was distributed above equilibrium across both membranes, consistent with active accumulation of Cl-. A sixfold decrease in Cl- in the basal bath depolarized Vba by 12 +/- 1 mV (n = 17) and increased the apparent basal membrane resistance. By sequential measurement of aiCl and subepithelial Cl- activity during a step decrease in basal Cl-, we constructed the change in Cl- equilibrium potential (ECl) across the basal membrane. Estimation of the change in basal membrane electromotive force during the change in ECl gave an average value for the Cl- transference number (TCl) of 0.45. Further evidence for a Cl- conductance was obtained by measuring changes in aiCl induced by transepithelial current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. Both the amplitude of the Cl- diffusion potential and the current-induced changes in aiCl were reduced by basal perfusion with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250-500 microM), a blocker of Cl- channels in some epithelia.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Investigation of the effects of beta-2 stimulation on free fatty acids in man.
In this study we present evidence that lipolysis in man is under beta-2 adrenergic control and that beta-2 stimulation produces a characteristic profile of individual free fatty acid (FFA) release. Twelve healthy volunteers received infusions of placebo (N Saline), terbutaline (a selective beta-2 agonist) and dilevalol (a new non-selective beta-blocker with beta-2 agonist activity). Plasma FFA concentrations during and after the infusions were measured using gas chromatography. A significant rise in total and individual FFAs was seen after 30 min of terbutaline infusion. This was most marked for oleic acid. Total and individual FFA concentrations also rose after 30 min of dilevalol infusion; this was only significant for oleic acid and was approximately 15% of the rise induced by terbutaline infusion. Placebo infusion did not cause any significant changes in FFA levels.
Adult↗
Effects of current clamp on chick retinal pigment epithelium.
The basal membrane of the retinal pigment epithelium (RPE) is the origin of two components of the electroretinogram, the fast oscillation and the light peak. Both of these responses originate from changes in basal membrane potential (Vba), and both are associated with changes in basal membrane resistance (Rba). In addition, many experimental manipulations that alter Vba also produce apparent changes in Rba. These findings raise the possibility that the basal membrane contains a voltage-sensitive conductance that operates in the physiologic range and is involved causally in light-evoked and other responses. We report the results of current clamp experiments on the isolated retina-RPE-choroid of chick that were designed to test for the presence of such a voltage-sensitive conductance in the basal membrane. Depolarizing Vba by 15 mV with retina-to-choroid current had essentially no effect on either the ratio of membrane resistances (Rap/Rba) or the transtissue resistance (RTotal), indicating no alteration in Rba. In contrast, hyperpolarizing Vba by 15 mV with choroid-to-retina current caused a gradual decrease in RTotal and increase in Rap/Rba. Analysis of accompanying changes in membrane voltages and changes in intracellular c-wave amplitude suggested that the most likely cause of the decrease in RTotal is a decrease in paracellular resistance. Voltage-sensitive conductances of the basal membrane appear to play little or no role in the resistance changes that accompany changes in Vba in the physiologic range. The conductance changes underlying the fast oscillation and light peak probably result from either the modulation of channels by second messengers or changes in intracellular ion concentration.
Animals↗
Voltage-dependent currents in isolated cells of the frog retinal pigment epithelium.
1. Retinal pigment epithelial (RPE) cells were isolated enzymatically from bullfrog retinae. The patch-clamp technique was employed to investigate whole-cell currents under voltage-clamp conditions. 2. Isolated RPE cells were columnar or cuboidal in form, often with long processes protruding from the apical surface. Distinct apical and basal membrane domains were maintained for several hours following isolation. 3. The mean membrane capacitance was 62 pF. The resting potential averaged -30 mV, but it was as high as -75 mV in some cells. 4. Three voltage-dependent currents were observed: a time-independent and inwardly rectifying current and two time-dependent outwardly rectifying currents that had distinct kinetic properties. 5. Voltage pulses from a holding potential of -70 mV to potentials ranging from -30 to -120 mV produced membrane currents that were essentially time independent. The I-V relationship in this voltage range depended on the resting potential. It was usually inwardly rectifying in cells with resting potentials negative to about -50 mV, but tended to be linear in cells with more positive potentials. Three observations strongly suggested that the inwardly rectifying current is carried by K+. First, increasing the extracellular K+ concentration [( K+]) from 2 to 112 mM shifted the zero-current potential of the I-V relationship in the positive direction from an average value of -60 mV to 0 mV. Second, the addition of the K+ channel blockers Ba2+ (2 mM) or Cs+ (5 mM) to the extracellular solution inhibited a major component of the inwardly rectifying current. Finally, the reversal potential (Vr) of the Ba2(+)-sensitive current averaged -90 mV, near the K+ equilibrium potential (EK). 6. In approximately 50% of the cells, depolarizing voltage pulses to potentials more negative than -30 mV evoked an outward current that resembled the delayed rectifier present in other non-excitable cells. It activated with sigmoidal kinetics in less than 100 ms following a brief delay and then declined exponentially with a time constant of approximately 1 s. The peak chord conductance associated with this current was half-maximal at +14 mV. Several observations indicated that this outwardly rectifying current is carried primarily by K+: its Vr closely matched EK over a wide range of extracellular [K+]; it was inhibited 80% by exposure to the K+ channel blockers 4-aminopyridine (1 mM) and tetraethylammonium (20 mM); and it was abolished by intracellular dialysis with a K(+)-free solution.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Investigation of the beta 2-agonist properties of dilevalol: metabolic effects of intravenous infusion.
We describe two studies which investigate the beta 2-agonist properties of dilevalol. We have previously demonstrated, by giving an infusion of terbutaline to human volunteers, that beta 2-stimulation causes a rise in plasma glucose and a fall in plasma potassium. These metabolic effects can be prevented by prior beta-blockade. We now show that infusion of dilevalol produces similar, but quantitatively smaller, metabolic effects at a dose which also produces clinically significant beta-blockade, as judged by a fall in exercise heart rate. This adds support to the claim that dilevalol is a non-selective beta-blocker with selective beta 2-agonist activity.
Adult↗
Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium.
Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
cAMP stimulates the Na+-K+ pump in frog retinal pigment epithelium.
Adenosine 3', 5'-cyclic monophosphate (cAMP) induced increases in active Na+ secretion and K+ absorption that were blocked by apical ouabain (10(-4) M), suggesting stimulation of the Na+-K+ pump. cAMP also produced rapid membrane voltage and resistance changes that could be divided chronologically into three phases. In phase 1, the basolateral membrane depolarized at a faster rate than the apical membrane, probably as a result of an increase in basolateral membrane conductance. In phase 2, the apical membrane repolarized toward control faster than the basal membrane, whereas in phase 3 the basolateral membrane repolarized faster than the apical membrane. Apical ouabain completely inhibited the cAMP-induced repolarization of the apical membrane during phase 2. Thus the stimulation of the Na+-K+ pump occurs within minutes of cAMP elevation. Na+ removal from the basal side did not block the cAMP-induced voltage changes, indicating that the initial conductance increase is not due to Na+. In contrast, Na+ removal from the apical bath inhibited all phases of the cAMP response. This suggests that apical membrane Na+-dependent transport mechanisms mediate the stimulation of the Na+-K+ pump. cAMP also caused a significant drop in intracellular K+ activity (approximately 5 mM) that preceded phase 2. This drop could stimulate the Na+-K+ pump, as suggested by previous experiments.
Animals↗
Adenylate cyclase stimulation alters transport in frog retinal pigment epithelium.
The effect of adenylate cyclase inhibitors and activators on enzyme kinetics and cyclic AMP (cAMP) levels was determined in the bullfrog retinal pigment epithelium (RPE). The RPE enzyme has two Km for ATP: 5.2 X 10(-4) and 4.0 X 10(-5) M, with Vmax of 2.5 and 0.25 nmol X mg protein-1 X min-1. Forskolin, the most potent activator, produced a fourfold increase in enzyme activity and, in the presence of isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterase, caused a 20-fold increase in cAMP levels. Alloxan, a potent inhibitor, blocked the forskolin-induced activation of this enzyme. In the isolated RPE choroid, forskolin (plus IBMX) produced changes in membrane voltage and resistance that were similar in magnitude but slower in time course than those produced by exogenous cAMP. Like exogenous cAMP, forskolin also decreased steady-state fluid and solute transport in isotonic proportions. Therefore, modulation of RPE adenylate cyclase activity plays an important role in the control of RPE transport.
1-Methyl-3-isobutylxanthine↗
Effects of cyclic AMP on fluid absorption and ion transport across frog retinal pigment epithelium. Measurements in the open-circuit state.
A modified version of a capacitance probe technique has been used to measure fluid transport across the isolated retinal pigment epithelium (RPE)-choroid of the bullfrog. The accuracy of this measurement is 0.5-1.0 nl/min. Experiments carried out in the absence of external osmotic or hydrostatic gradients show that the RPE-choroid transports fluid from the retinal to the choroid side of the tissue at a rate of approximately 10 nl/min (4-6 microliters/cm2 X h). Net fluid absorption (Jv) was abolished within 10 min by the mitochondrial uncoupler 2,4-dinitrophenol. It was also inhibited (70%) by the removal of bicarbonate from the bulk solutions bathing the tissue. Ouabain caused a slow decrease in Jv (no effect at 10 min, 70% at 3 h), which indicates that RPE fluid transport is not directly coupled to the activity of the Na-K pump located at the apical membrane of this epithelium. In contrast to ouabain, cyclic AMP (cAMP) produced a quick decrease in Jv (84% within 5 min). Radioisotope experiments in the open circuit show that cAMP stimulated secretory fluxes of Na and Cl, which accounted for the observed cAMP-induced decrease in Jv. The direction of net fluid absorption, the magnitudes of the net ionic fluxes in the open circuit, and the dependence of Jv on external bicarbonate concentration strongly suggest that fluid absorption is generated primarily by the active absorption of bicarbonate.
Absorption↗
Regeneration of beta-adrenergic receptors in senescent rats: a study using an irreversible binding antagonist.
The drug used in this study, bromoacetylalprenololmenthane, has the ability to bind and block irreversibly beta-adrenergic receptors. The drug was bound to membranes prepared from hearts, lungs, and brains of both senescent and young rats with a similar affinity. When this drug was injected into rats in nontoxic doses (up to 70 mg/kg), up to 90% of beta-adrenergic receptors were irreversibly blocked 4 hr after injection, whereas the injection of similar amounts of (+/-)-alprenolol was without effect on receptor number. In senescent animals this blockade lasted considerably longer than in young animals; receptor numbers in hearts and lungs of senescent rats returned to control levels only 1 month after injection. The number of beta-adrenergic receptors in brains of senescent rats was unaffected by this drug. Thus, based upon the long-lasting blockade of beta-adrenergic receptors in therapeutically important organs, it appears that irreversible binding blockers may have potential in the treatments of senescent organisms.
Adrenergic beta-Antagonists↗
Fluid transport across retinal pigment epithelium is inhibited by cyclic AMP.
Fluid transport across the retinal pigment epithelium (bullfrog) has been measured. These experiments were carried out by using a capacitance probe technique and a water-impermeable chamber that allowed the measurements to be made with an accuracy of 0.5-1.0 nl/min. With identical Ringer's solution on both sides of the epithelium, and in the absence of a hydrostatic driving force, the direction of net fluid movement is from the retina to the choroid (absorption). The net transport rate, approximately 10 nl/min (4.8 microliters/cm2 . hr), is comparable to that observed in other amphibian epithelia. It is reduced to zero by the mitochondrial uncoupler 2,4-dinitrophenol but is relatively unaffected by ouabain, which inhibits the Na+/K+-pump located on the apical membrane of this epithelium. A significant decrease in net fluid absorption was produced by dibutyryl cAMP and 3-isobutyl-1-methylxanthine (a potent phosphodiesterase inhibitor). This cAMP-dependent fluid transport may be an important mechanism for controlling the fluid volume in the subretinal space.
1-Methyl-3-isobutylxanthine↗
Age-related decrease in repair of oxidative damage to surface sulfhydryl groups on rat adipocytes.
Adipocytes were isolated from the Wistar and Sprague-Dawley strains of rats of different ages. An impermeable reagent, mercury-[3H] dextran, was used to quantitate the sulfhydryl groups on the surface of these cells. The application of this reagent after the pre-reduction of cells with 2-mercaptoethanol yielded a measure of the total sulfhydryl and disulfide groups located there. Aging was found to significantly decrease the ratio of the number of exofacial sulfhydryl groups to the total number of exofacial sulfhydryl and disulfide groups.
Adipose Tissue↗