Biomedical subjects

D A Carré

Publications and source records attributed to D A Carré.

15 recordsLinked to original sources

PubMed2001

Timolol may inhibit aqueous humor secretion by cAMP-independent action on ciliary epithelial cells.

The beta-adrenergic antagonist timolol reduces ciliary epithelial secretion in glaucomatous patients. Whether inhibition is mediated by reducing cAMP is unknown. Elemental composition of rabbit ciliary epithelium was studied by electron probe X-ray microanalysis. Volume of cultured bovine pigmented ciliary epithelial (PE) cells was measured by electronic cell sizing; Ca(2+) activity and pH were monitored with fura 2 and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, respectively. Timolol (10 microM) produced similar K and Cl losses from ciliary epithelia in HCO/CO(2) solution but had no effect in HCO/CO(2)-free solution or in HCO/CO(2) solution containing the carbonic anhydrase inhibitor acetazolamide. Inhibition of Na(+)/H(+) exchange by dimethylamiloride in HCO/CO(2) solution reduced Cl and K comparably to timolol. cAMP did not reverse timolol's effects. Timolol (100 nM, 10 microM) and levobunolol (10 microM) produced cAMP-independent inhibition of the regulatory volume increase (RVI) in PE cells and increased intracellular Ca(2+) and pH. Increasing Ca(2+) with ionomycin also blocked the RVI. The results document a previously unrecognized cAMP-independent transport effect of timolol. Inhibition of Cl(-)/HCO exchange may mediate timolol's inhibition of aqueous humor formation.

Animals↗

PubMed2001

Reliable measurement of mouse intraocular pressure by a servo-null micropipette system.

PURPOSE: To develop a reliable technique for measuring intraocular pressure (IOP) in the mouse. METHODS: An electrophysiologic approach-the servo-null micropipette system (SNMS)-for measuring hydrostatic pressure was adapted for the mouse eye. Fine-tipped (5 microm in diameter) micropipettes were advanced across the cornea with a piezoelectric micromanipulator, and the IOP was continuously monitored for up to 46 minutes. RESULTS: The micropipette tip was visualized in the anterior chamber. With the SNMS, the IOP of black Swiss outbred mice under ketamine anesthesia was 17.8 +/- 0.4 mm Hg, higher than values previously estimated in inbred mouse strains by a larger bore microneedle manometric technique. After withdrawal of the micropipette, a second penetration led to a similar level of IOP. Hypotonic solutions increased and hypertonic solutions decreased IOP. Drugs that decrease inflow (acetazolamide, timolol) or increase outflow facility (pilocarpine, latanoprost) in primates and humans lowered steady state IOP in the mouse. The transient initial increase in IOP produced by pilocarpine reported in other animals was also observed in the mouse. Xylazine-ketamine anesthesia lowered IOP substantially in comparison with systemic anesthesia with either ketamine or tribromoethanol alone. CONCLUSIONS: The SNMS is the first reliable, reproducible method for measuring mouse IOP. The mouse IOP is sensitive not only to drugs known to reduce aqueous humor inflow but also to drugs that increase aqueous humor outflow facility in the eyes of primates and humans. The development of the SNMS is an enabling step in the use of the mouse for glaucoma research, including molecular genetics, molecular pharmacology, and the search for novel antiglaucoma drugs.

Acetazolamide↗

PubMed2000

Similarity of A(3)-adenosine and swelling-activated Cl(-) channels in nonpigmented ciliary epithelial cells.

Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl(-) transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A(3)-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)-N-methyl-be ta-D-ribofuranuronamide (IB-MECA) stimulated currents (I(IB-MECA)) by approximately 90% at +80 mV. Partial replacement of external Cl(-) with aspartate reduced outward currents and shifted the reversal potential (V(rev)) from -23 +/- 2 mV to -0.0 +/- 0.7 mV. Nitrate substitution had little effect. Perfusion with the Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid inhibited the currents. Partial Cl(-) replacement with aspartate and NO(3)(-), and perfusion with NPPB, had similar effects on the swelling-activated whole cell currents (I(Swell)). Partial cyclamate substitution for external Cl(-) inhibited inward and outward currents of both I(IB-MECA) and I(Swell). Both sets of currents also showed outward rectification and inactivation at large depolarizing potentials. The results are consistent with the concept that A(3)-subtype adenosine agonists and swelling activate a common population of Cl(-) channels.

Adenosine↗

PubMed1999

A3 adenosine receptors regulate Cl- channels of nonpigmented ciliary epithelial cells.

Adenosine stimulates Cl- channels of the nonpigmented (NPE) cells of the ciliary epithelium. We sought to identify the specific adenosine receptors mediating this action. Cl- channel activity in immortalized human (HCE) NPE cells was determined by monitoring cell volume in isotonic suspensions with the cationic ionophore gramicidin present. The A3-selective agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) triggered shrinkage (apparent Kd = 55 +/- 10 nM). A3-selective antagonists blocked IB-MECA-triggered shrinkage, and A3-antagonists (MRS-1097, MRS-1191, and MRS-1523) also abolished shrinkage produced by 10 microM adenosine when all four known receptor subtypes are occupied. The A1-selective agonist N6-cyclopentyladenosine exerted a small effect at 100 nM but not at higher or lower concentrations. The A2A agonist CGS-21680 triggered shrinkage only at high concentration (3 microM), an effect blocked by MRS-1191. IB-MECA increased intracellular Ca2+ in HCE cells and also stimulated short-circuit current across rabbit ciliary epithelium. A3 message was detected in both HCE cells and rabbit ciliary processes using RT-PCR. We conclude that human HCE cells and rabbit ciliary processes possess A3 receptors and that adenosine can activate Cl- channels in NPE cells by stimulating these A3 receptors.

Adenosine↗

PubMed1998

A release mechanism for stored ATP in ocular ciliary epithelial cells.

Purines can modify ciliary epithelial secretion of aqueous humor into the eye. The source of the purinergic agonists acting in the ciliary epithelium, as in many epithelial tissues, is unknown. We found that the fluorescent ATP marker quinacrine stained rabbit and bovine ciliary epithelia but not the nerve fibers in the ciliary bodies. Cultured bovine pigmented and nonpigmented ciliary epithelial cells also stained intensely when incubated with quinacrine. Hypotonic stimulation of cultured epithelial cells increased the extracellular ATP concentration by 3-fold; this measurement underestimates actual release as the cells also displayed ecto-ATPase activity. The hypotonically triggered increase in ATP was inhibited by the Cl--channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in both cell types. In contrast, the P-glycoprotein inhibitors tamoxifen and verapamil and the cystic fibrosis transmembrane conductance regulator (CFTR) blockers glybenclamide and diphenylamine-2-carboxylate did not affect ATP release from either cell type. This pharmacological profile suggests that ATP release is not restricted to P-glycoprotein or the cystic fibrosis transmembrane conductance regulator, but can proceed through a route sensitive to NPPB. ATP release also was triggered by ionomycin through a different NPPB-insensitive mechanism, inhibitable by the calcium/calmodulin-activated kinase II inhibitor KN-62. Thus, both layers of the ciliary epithelium store and release ATP, and purines likely modulate aqueous humor flow by paracrine and/or autocrine mechanisms within the two cell layers of this epithelium.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Effects of HCO3- on cell composition of rabbit ciliary epithelium: a new model for aqueous humor secretion.

PURPOSE: To determine whether the Na+-K+-2Cl- symport or the parallel Na+/H+ and Cl-/HCO3- antiports provide the dominant pathway for NaCl uptake into the ciliary epithelium. Both pathways are known to support NaCl entry from the stroma into the pigmented ciliary epithelial (PE) cells, after which Na+ and Cl- diffuse across the gap junctions into the nonpigmented ciliary epithelial (NPE) cells and are released into the aqueous humor. METHODS: Rabbit iris ciliary bodies were preincubated in HCO3-/CO2-containing or HCO3-/CO2-free solutions before quick freezing, cryosectioning, dehydration, and electron probe x-ray microanalysis. RESULTS: The NPE and the PE cells contained more K and Cl when incubated with bicarbonate. Inhibition of carbonic anhydrase with 0.5 mM acetazolamide had little effect in HCO3--free medium but prevented the increase in Cl in both cell types in HCO3-/CO2 solution. Inhibition of the Na+-K+-2Cl- symport with 10 to 500 microM bumetanide caused Cl loss from both cell types in HCO3--free solution, but bumetanide produced a paradoxical increase in Cl and Na in HCO3-/CO2 solution. Together, acetazolamide and bumetanide resulted in significant Cl loss in HCO3--free solution and prevented the gains of Cl and Na in HCO3-/CO2 solution. CONCLUSIONS: The present results indicate that the dominant entry pathway of NaCl from the stroma into the ciliary epithelial syncytium is through an acetazolamide-inhibitable Cl-/HCO3 and a parallel Na+/H+ antiport. The dominant release pathways into the aqueous humor appear to be a Na+-K+-2Cl-symport, which can be outwardly directed under physiological conditions, together with the Na+/K+-exchange pumps and Cl- channels.

Animals↗

PubMed1997

Adenosine stimulates Cl- channels of nonpigmented ciliary epithelial cells.

Ciliary epithelial cells possess multiple purinergic receptors, and occupancy of A1 and A2 adenosine receptors is associated with opposing effects on intraocular pressure. Aqueous adenosine produced increases in short-circuit current across rabbit ciliary epithelium, blocked by removing Cl- and enhanced by aqueous Ba2+. Adenosine's actions were further studied with nonpigmented ciliary epithelial (NPE) cells from continuous human HCE and ODM lines and freshly dissected bovine cells. With gramicidin present, adenosine (> or = 3 microM) triggered isosmotic shrinkage of the human NPE cells, which was inhibited by the Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) and niflumic acid. At 10 microM, the nonmetabolizable analog 2-chloroadenosine and AMP also produced shrinkage, but not inosine, UTP, or ATP. 2-Chloroadenosine (> or = 1 microM) triggered increases of whole cell currents in HCE cells, which were partially reversible, Cl- dependent, and reversibly inhibited by NPPB. Adenosine (> or = 10 microM) also stimulated whole cell currents in bovine NPE cells. We conclude that occupancy of adenosine receptors stimulates Cl- secretion in mammalian NPE cells.

2-Chloroadenosine↗

PubMed1996

Electron probe X-ray microanalysis of rabbit ciliary epithelium.

Rabbit iris-ciliary bodies were preincubated in control and experimental Ringer's solutions before quick freezing, cryosectioning, dehydration and electron probe X-ray microanalysis. After preincubation in a baseline bicarbonate-free Cl- Ringer's solution, the ciliary epithelial intracellular Na+, K+ and Cl- concentrations were estimated to be 15 +/- 3, 162 +/- 14 and 46 +/- 5 mmol kg-1 intracellular water, respectively. The water and elemental Na, K, Cl and P contents were similar in the non-pigmented (NPE) and pigmented (PE) ciliary epithelial cells. As expected, inhibition of the Na,K-exchange pump by preincubation with ouabain markedly increased the intracellular Na content, and markedly reduced the intracellular K content, verifying the validity of the experimental analysis. The Cl- channels of the NPE cells likely play a critical role in determining the rate of aqueous humor formation. Therefore, we have examined the effects of altering Cl- transport on the intracellular composition in this initial microprobe study of the ciliary epithelium. As expected, exposure to bicarbonate increased the intracellular Cl and water contents. Replacement of external Cl- by NO3- was twice as effective as replacement by gluconate in leaching Cl- out of the intracellular compartment. An unexpected finding was that NO3- replacement of internal Cl- substantially increased the intracellular Na and decreased the intracellular K content, possibly by stabilizing the Na,K-pump in the E1P form and inhibiting enzyme activity.

Animals↗

PubMed1996

Cell-attached patch clamping of the intact rabbit ciliary epithelium.

Following thorough removal of adhering aqueous humor, we have succeeded in patch clamping the intact rabbit ciliary epithelium in the cell-attached and inside-out excised-patch modes. Rapidly fluctuating currents ("chatter activity') were observed during recordings conducted for as long as 1 h. Chatter activity did not reflect seal instability since interconversion was noted between chatter activity and transitions between stable open and closed states, excision of patches into the bath was associated with substantial shifts in the reversal potential, and chatter activity could be triggered by sustained hyperpolarization, but was insensitive to stretch. The chatter channel was identified as cation-nonselective from the reversal potentials both in the cell-attached and excised-patch modes. The channel's kinetics were similar to those of the cGMP-activated phototransduction channel. The results of PCR amplifications of fragments in cDNA libraries from both human ciliary body and human nonpigmented ciliary epithelial (NPE) cells indicated that human ciliary epithelial cells transcribe message for the retinal phototransduction channel. The possible role of the phototransduction channel in expressing chatter activity was further explored by perfusing preparations with a known activator of that channel (cGMP) and with a known inhibitor (L-cis-diltiazem). Neither agent significantly affected chatter behavior. We conclude that: (1) this is the first demonstration of the feasibility of patch-clamping the intact ciliary epithelium; (2) the NPE cells display chatter activity arising from rapidly fluctuating transitions of a cation-nonselective channel; (3) NPE cells can transcribe message for the cation-nonselective phototransduction channel; and (4) if the observed chatter activity is from a homologue of the photo-transduction channel, the homologue is pharmacologically distinct.

Animals↗

PubMed1995

cGMP modulates transport across the ciliary epithelium.

cGMP reduced the short-circuit current (ISC) when applied to the aqueous surface of isolated rabbit and cat ciliary epithelia. cGMP either stimulated (in the rabbit) or had no effect (in the cat) on ISC when applied to the stromal surface. Addition of the cGMP-mediated hormone atrial natriuretic peptide (ANP) to the stromal (but not the aqueous) surface, or the nitrovasodilator sodium nitroprusside to the stromal surface, inhibited ISC across rabbit ciliary epithelium. The response to stromal cGMP was partly mediated by K+ channels at the stromal surface of the rabbit pigmented epithelial (PE) cells, since the effect was inhibited by stromal Ba2+, and was unaffected by Cl- replacement, by bumetanide, or by DIDS. In contrast, the response to aqueous cGMP was not likely mediated by changing either K+ or Cl- channels, based on transepithelial measurements of rabbit ciliary epithelium and complementary whole-cell patch clamping of cultured human nonpigmented ciliary epithelial (NPE) cells. The possibility of interacting effects between cGMP and cAMP in targeting the Na+, K(+)-exchange pump was also considered. Strophanthidin blocked the responses to either aqueous or stromal cGAMP. Applying 10 microns forskolin to generate endogenous cAMP enhanced the subsequent response to aqueous cGMP by approximately equal to 80%. We conclude that cGMP has at least two actions on the ciliary epithelium. The major effect may be to reverse cAMP-mediated inhibition of the NPE Na+ pumps at the aqueous surface of both rabbit and cat ciliary epithelia. The second effect is likely mediated by increasing K(+)-channel and pump activity of the rabbit PE cells at the stromal surface.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1992

Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling.

Anisosmotic cell swelling triggers a regulatory volume decrease (RVD) in cell lines derived from human nonpigmented ciliary epithelium. Measurements of cell volume have indicated that the RVD reflects activation of K+ and/or Cl- channels. We have begun to characterize the putative channels by whole cell patch clamping. The results obtained by altering the external K+ and Cl- concentrations and by adding 20-50 microM quinidine or 1 mM Ba2+ indicate that K+ conductances contribute substantially and Cl- conductances contribute very little to the total membrane conductance (GT) under baseline isotonic conditions. Reducing the external osmolality by 20-50% reversibly and reproducibly increased GT by an order of magnitude. Data obtained from ion substitutions and the channel blockers quinidine and 5-nitro-2-(3-phenylpropylamino)-benzoate indicate that most of the hypotonicity-induced conductance reflects stationary Cl(-)-channel activity. The contribution of new K(+)-channel activity was small at intracellular free Ca2+ concentrations of 10 or 200 nM. We conclude that the RVD triggered by bath hypotonicity primarily reflects increased Cl(-)-channel activity.

Chlorides↗

PubMed1991

Effects of adrenergic agents on transepithelial electrical measurements across the isolated iris-ciliary body.

Transmembrane electrical measurements were performed on the isolated rabbit iris-ciliary body to study direct effects of adrenergic drugs on the ciliary epithelium. Alpha-adrenergic agonists (epinephrine, norepinephrine, or phenylephrine) lowered the short-circuit current (SCC) in a dose-dependent fashion relative to which chamber side the drug was added: simultaneous addition to both chambers greater than blood side only greater than aqueous side only. Pretreatment (5 x 10(-5) M) with the non-selective beta-adrenergic antagonist timolol had no effect while the non-selective alpha-adrenergic antagonist, phentolamine, completely prevented the alpha agonist-induced decrease in SCC. The alpha-adrenergic response was mediated by the alpha 1 subtype since prazosin, but not yohimbine, blocked the induced reduction in SCC. The beta-adrenergic agonist isoproterenol caused a dose-dependent decrease in the SCC. The decrease was similar when the drug was added to only the blood side or to both sides of the chamber. Addition to only the aqueous chamber had no effect. Pretreatment with beta-adrenergic antagonists blocked the isoproterenol response: non-selective = selective beta 2 greater than selective beta 1. The isoproterenol-induced decrease in SCC was also blocked by non-selective alpha-adrenergic antagonists. The response was mediated by the alpha 1 subtype since prazosin, but not yohimbine, blocked the isoproterenol response. This suggests that isoproterenol interacted with the alpha 1-adrenergic sensitive pathway in the rabbit ciliary process.

Animals↗

PubMed1991

Halogenated inhalation anesthetic agents decrease transepithelial electrical measurements across the isolated iris-ciliary body.

Transmembrane electrical measurements were performed on the isolated rabbit iris-ciliary body (I-CB) to study the direct effects of halogenated inhalation anesthetic agents on the ciliary epithelium. Addition of either halothane, enflurane, or isoflurane to the control 95% O2:5% CO2 gas mixture resulted in a dose-dependent decrease in the short-circuit current (SCC) and potential difference (PD). This response was reversible after the anesthetic gas was discontinued. Pretreatment with either alpha-adrenergic or beta-adrenergic antagonists (phentolamine or timolol) had no effect on the halothane-induced decrease in SCC. Delivery of the analgestic gas N2O did not alter baseline electrical measurements across the isolated I-CB.

Analysis of Variance↗

PubMed1990

Optic disc topography in patients with low-tension and primary open angle glaucoma.

Computerized optic disc topographic measurements (cup-disc ratio, neuroretinal rim area, disc volume) and automated visual field parameters (total field loss, mean sensitivity) were similar in 48 eyes with primary open angle glaucoma and 27 eyes with low-tension glaucoma. In open angle glaucoma, the neuroretinal rim area was negatively correlated with the cup-disc ratio and the disc volume. Total field loss was positively correlated with the cup-disc ratio and the disc volume and negatively correlated with the neuroretinal rim area. Eyes with low-tension glaucoma showed a negative correlation between neuroretinal rim area and cup-disc ratio, and between total field loss and rim area. However, low-tension glaucomatous eyes did not have significant correlations between disc volume and rim area and between total field loss and cup-disc ratio or disc volume. Cupping in low-tension glaucoma was broadly sloping, resulting in less disc volume alteration.

Aged↗

PubMedDate not supplied

Formation of the aqueous humor.

1. Glaucoma is a worldwide disease affecting approximately 1-2% of the population aged over 35 years in industrial countries and is a major cause of blindness. 2. Glaucoma is usually associated with an increased intraocular pressure reflecting an imbalance between the rate of production of fluid (the aqueous humor) by the ciliary epithelial cells and its drainage from the eye. Therefore, it is important to understand how this secretion is produced. This requires a knowledge of ciliary epithelial cell composition, which has, in the past, proved difficult to obtain in mammalian preparations. 3. We have recently used the technique of electron-probe X-ray microanalysis to determine this composition under a variety of in vitro conditions. 4. Our results have led to a new model for this secretion that emphasizes the potential secretory role of the Na+/K+/2Cl- cotransporter.

Animals↗