Susanne Berweck

Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells

Endothelin-like immunoreactivity was detected in human (15.6 +/- 2.7 pg/ml) and bovine (11.1 +/- 0.98 pg/ml) aqueous humour of the eye. These concentrations are 2-3 times higher than the corresponding plasma levels. Cultured human nonpigmented ciliary epithelial cells released endothelin-like immunoreactivity with a maximum of 2.1 +/- 0.32 pg/(cm2* 48 h). The release was stimulated by fetal calf serum, thrombin, carbachol and phorbol ester and blocked by cycloheximide. Immunocytochemistry showed cytoplasmic staining of cultured human nonpigmented ciliary epithelial cells for endothelin-1. Endothelin-1 was shown to induce contractions in isolated human ciliary muscle by isometric force measurements. Endothelin in the aqueous humour may play a role in the regulation of intraocular pressure.

Characterization of Cl−HCO3− exchange in cultured bovine pigmented ciliary epithelium

Many recent data indicate that transport of Cl- across the ciliary epithelium plays an important role in aqueous humor formation. We used 36Cl to investigate the pathways for Cl- transport in confluent monolayers of cultured bovine pigmented ciliary epithelial cells. Cl- uptake mainly occurred via a mechanism with typical characteristics of an anion exchanger, and could be stimulated by an outwardly directed HCO3- gradient. One mM SITS and 1 mM DIDS inhibited Cl- uptake by some 80-90%, the latter with an IC50 of about 20 microM. HCO3- stimulated Cl- uptake could be partly inhibited for furosemide and to a lesser extent by bumetanide, indicating an action of loop-diuretics on the anion exchanger. 36Cl- uptake was cis-inhibited by the halides Cl-, I- and Br-, by NO3-, formate and acetate. Inhibition of Cl- uptake by extracellular HCO3- was less effective in the absence of extracellular Na+, suggesting that not only HCO3- but also NaCO3- binds to the carrier. SO2/4-, cyclamate and gluconate did not significantly reduce Cl- uptake via the anion exchanger. DIDS-senstive Cl- uptake showed saturation kinetics with respect to the Cl- concentration with an apparent Km of 8 mM. Cl- efflux could be stimulated by external Cl- and HCO3- and was inhibited by DIDS. Thus, cultured bovine pigmented ciliary epithelial cells express a Cl-/HCO3- exchanger. A possible role of this carrier system for aqueous humor formation is discussed [corrected].

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells

Uptake studies with22Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na+ transport. A large part of Na+ uptake was sensitive to amiloride, quinidine and harmaline. Na+ uptake was stimulated by intracellular acidification (using the NH4+ prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentKM for Na+ from 38 to 86 mM without considerable changes inVmax. In the presence of 5 mM Na+ half maximal inhibition of amiloride sensitive Na+ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na+ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H+ seemed to be competitive with aKi of 20–40 nM. 10 μM amiloride increased the apparentKM for Na+ from 33 mM to 107 mM, whileVmax remained nearly unchanged. IC50 for amiloride was 6 μM at 5 mM Na+ and 36 μM in the presence of 150 mM Na+. Thus, amiloride behaves as a competitive inhibitor with aKi of about 5 μM. The affinities of Na+ to the transport site (KM≈16 mM), to the inhibitory site for protons (KM≈21 mM), and to the inhibitory site for amiloride (KM≈26 mM) were in the same order of magnitude.In summary, we have presented evidence for the presence of a Na+/H+ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na+, H+ and amiloride.

Large conductance calcium-activated potassium channels in cultured retinal pericytes under normal and high-glucose conditions

Pericytes are considered to contribute to the regulation of retinal microcirculation which is impaired in diabetic retinopathy. Single, large-conductance, Ca2+-dependent K+ channels (BK) were studied in cultured bovine retinal capillary pericytes using the patch-clamp method. In excised patches with symmetrical 135-mmol/l K+ solutions a single channel conductance of 238±9.9 pS was measured. With a K+ gradient of 4/ 135 mmol/l (extracellular/intracellular) the slope conductance averaged 148±2.9 pS at 0 mV. The mean permeability was 4.2×10−13 cm3/s. The channel was highly selective for K+ with a permeability ratio for K+ over Na+ of 1/0.02. The mean open time and the open probability (Po) of the BK channel increased with depolarization and with increasing internal [Ca2+] showing a maximal sensitivity to Ca2+ between 10−4 and 10−5 mol/l Ca2+. Ba2+ (5 mmol/l), quinine (5 mmol/l), and verapamil (Michaelis constant 1.5×10−5 mol/l) blocked from the intracellular side. Tetraethylammonium induced a dose-dependent block from the outside only with a halfmaximal blocking concentration of 2.5×10−4 mol/l. Charybdotoxin (10−8 mol/l) blocked completely from the extracellular side. The channel activity was not changed by either internal adenosine triphosphate (ATP, 10−4 mol/l) or the putative opener of ATP-sensitive K+ channels Hoe 234 (10−6 mol/l). In cell-attached patches channelPo was less than 3%. After a 3-day incubation in culture medium containing an elevated glucose concentration (22.5 mmol/l) the channel activity in attached patches was markedly increased. These data indicate that cultured retinal pericytes possess a BK channel. The activity of the channel increases after incubation with elevated glucose concentrations, which could indicate altered regulation of the channel under these conditions. The implications of altered function of BK channels are discussed with respect to haemodynamic changes observed in diabetic retinopathy.

Characterization of acetylcholine- and endothelin-induced calcium entry in cultured human ciliary muscle cells

We characterized the effects of acetylcholine and endothelin on cultured human ciliary muscle cells, using the calcium-sensitive dye fura-2 to measure intracellular calcium and intracellular microelectrodes to measure the membrane potential. Both agonists, endothelin and acetylcholine, had a typcial biphasic effect on the intracellular calcium concentration. Calcium peaked initially, because of its release from intracellular stores, and then reached a plateau, owing to entry of extracellular calcium. Endothelin-induced calcium entry was almost completely blocked by addition of extracellular La3+ (50 μmol/l) and Ni2+ (1 mmol/l). Acetylcholine-induced calcium entry was likewise almost completely abolished by La3+ and Ni2+. Both endothelin and acetylcholine led to an initial transient hyperpolarization with a subsequent depolarization. The hyperpolarization of the membrane potential had a time course similar to the initial calcium peak, while the depolarization occurred parallel to the calcium plateau. The depolarization induced by both agonists was reduced in the presence of La3+ and Ni2+. Verapamil (10 μmol/l) had no effect on either the calcium entry or the depolarization. Acetylcholine did not induce a [Ca2+]i peak when it was applied during the endothelin-induced [Ca2+]i plateau and vice versa. The [Ca2+]i plateau was not higher with concomitant than with single application of acetylcholine or endothelin. Thus, calcium entry and membrane depolarization induced by acetylcholine and endothelin seem to be mediated by a common La3+- and Ni2+-sensitive but verapamil-insensitive mechanism.

Effect of elevated glucose concentration on membrane voltage regulation in retinal capillary pericytes

The influence of elevated glucose concentration on resting membrane voltage, electrogenic Na+-K+-ATPase, and ATP-sensitive potassium channels (KATP channels) was studied in cultured bovine retinal capillary pericytes using conventional microelectrodes. The resting membrane voltage in cells grown in medium containing 5 mM glucose (control) averaged –27 ± 1.2 mV (mean ± SE, n = 26) and was not different from cells grown in medium containing 22.5 mM glucose (–26 1.2 mV, n = 26). Addition of ouabain (10−4 M), a specific inhibitor of the Na+-K+-ATPase, depolarized the membrane potential by 3.6 ± 0.4 mV (n = 10) in cells grown under control conditions and 0.7 ± 0.2 mV (n = 6) in cells grown under elevated glucose conditions. Thus, electrogenic activity of the Na+-K+-ATPase was significantly (P < 0.0001) reduced to 19% compared with control conditions. Electrogenic Na+-K+-ATPase activity could be partially restored (ouabain-induced depolarization ΔV = 2.0 ± 0.2 mV, n = 6) in cells grown with high glucose in the presence of the aldose reductase inhibitor tolrestat (10−5 M). The potassium channel opener Hoe 234 (10−6 M) induced membrane potential hyperpolarization in control cells (ΔV = 7.3 ± 1.2 mV, n = 13), which could be completely inhibited by the KATP channel blocker glibenclamide (10−7 M, n = 5). This indicates that pericytes possess KATP channels. The effect of KATP channels on membrane voltage was not significantly changed (P = 0.16) in cells cultured under high-glucose conditions (ΔV = 9.6 ± 2.0 mV, n = 6). Acute changes of glucose concentration did not affect the membrane voltage (n = 6). We conclude that high glucose concentrations alter the activity of the Na+-K+-ATPase in retinal pericytes via a mechanism involving the polyol metabolism. Therefore, hyperglycemia may alter regulation of membrane voltage and contractility of pericytes and, hence, the regulation of retinal microcirculation in diabetes. Altered microcirculation could be an important factor in the pathogenesis of diabetic retinopathy.