Biomedical subjects

M Wiederholt

Publications and source records attributed to M Wiederholt.

At least 37 records · Page 2Linked to original sources

PubMed1997

Effect of diuretics, channel modulators and signal interceptors on contractility of the trabecular meshwork.

Measurements of isometric tension were performed on isolated trabecular meshwork (TM) and ciliary muscle (CM) strips of the bovine eye. Anterior segments of bovine eyes with well-preserved TM were perfused to measure outflow rate. (1) Bumetanide (10(-4) mol/l) and hydrochlorothiazide (10(-4) mol/l) did not change the contractility of TM and CM strips in the presence or absence of carbachol. Ethacrynic acid dose-dependently relaxed TM and CM strips precontracted either by carbachol or endothelin. (2) Cytochalasin D totally relaxed TM and CM strips precontracted by carbachol. The outflow rate almost doubled after application of cytochalasin D. (3) The effects of various modulators of K+ and Ca2+ channels are summarized. Carbachol and endothelin are postulated to modify nonselective cation channels. An effective blocker of nonselective cation channels (flufenamic acid) relaxed TM and CM precontracted by carbachol or endothelin. These relaxing effects were independent of the relaxing effects of ethacrynic acid and isosorbide dinitrate. (4) Evidence for the presence of various transporters and receptors in TM cells is summarized. (5) The TM per se is a contractile element which is involved in the regulation of aqueous humor outflow. The contractility of TM and CM cells is differently modulated.

Absorption↗

PubMed1997

Anterior segment physiology after bumetanide inhibition of Na-K-Cl cotransport.

PURPOSE: To determine the effect of bumetanide inhibition of Na-K-Cl cotransport on aqueous humor formation and outflow facility in living cynomolgus monkeys, outflow facility in organ-cultured human eyes, and contraction in bovine ciliary muscle and trabecular meshwork strips, in vitro. METHODS: Aqueous humor formation in monkeys was measured fluorophotometrically for 6 hours, 1 to 6 weeks before, immediately after, and 2 to 6 weeks after bumetanide was administered intravitreally (final concentration approximately 100 or 500 microM) or intravenously (0.01 or 0.03 mg/ kg at 0 and 3 hours). Outflow facility in monkeys was determined by two-level, constant-pressure perfusion of the anterior chamber for 45 to 60 minutes before and after bumetanide was administered by bolus intracameral injection (100 microM, initial anterior chamber concentration) or by exchanging the anterior chamber with 2 ml 10, 100, or 500 microM bumetanide. Urine volume was measured 3 hours after administration of intravenous bumetanide in various diluents. The effect on intraocular pressure in organ-cultured human eyes was determined for 48 hours by constant-flow-variable-pressure perfusion with 10 microM bumetanide. Contraction of fresh bovine ciliary muscle and trabecular meshwork was measured isometrically with a force-length transducer system after exposure to 100 microM bumetanide +/-1 microM carbachol. RESULTS: The bumetanide concentrations used had little effect on outflow facility or on aqueous humor formation in normal monkeys, on intraocular pressure in organ-cultured human eyes, or on contraction of bovine ciliary muscle and trabecular meshwork strips. Intravenous bumetanide increased urine volume, regardless of the diluent used. CONCLUSIONS: These results suggest that Na-K-Cl cotransport is not involved functionally in regulation of aqueous humor inflow and outflow and in contractility of ciliary muscle and trabecular meshwork.

Aged↗

PubMed1997

Characterization of maxi-K-channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate.

PURPOSE: Electrophysiological characterization of trabecular meshwork cells and investigation of their response to elevation of cytosolic cyclic guanosine monophosphate (cGMP). METHODS: Bovine trabecular meshwork cells were cultured according to established methods and were studied, using the whole-cell and single-channel configurations of the patch-clamp technique. RESULTS: In single-channel experiments, cells expressed a channel with characteristics typical of maxi-K-channels. The channel was densely distributed in the membrane and had a high conductance of 326 +/- 4 pS (Pico Siemens) (symmetrical 150 mmol/l KCl; 37 degrees C) for potassium and negligible conductance for sodium (0.9 +/- 1 pS). The open probability could be elevated by depolarization, increasing cytosolic calcium, or adding adenosine triphosphate (1 mmol/ l). The channel could be blocked by external charybdotoxin (10(-8) mol/1), external TEA+ tetraethyl ammonium chloride (1 mmol/l) and by internal Ba2+ (10 mmol/l), whereas external Ba2+ and internal TEA+ (10 mmol/l) had no effect. In whole-cell experiments, trabecular meshwork cells displayed a strong outward conductance. Part of this conductance (35 +/- 5%) could be blocked by charybdotoxin and stimulated by ionomycin (10(-5) mol/1). Addition of 8-bromo-cGMP (10(-3) mol/1) stimulated the current to 290 +/- 57% (n = 4) of the original level, charybdotoxin led to a reduction of this current to 156 +/- 28% of the initial value. CONCLUSIONS: Trabecular meshwork cells express maxi-K-channels. These channels can be stimulated by raising internal cGMP levels and are known for their importance in smooth muscle relaxation. The results in this study supply further evidence that trabecular meshwork displays smooth muscle-like properties and contributes to the clarification of the mechanism leading to the relaxation of trabecular meshwork by nitrate and nonnitrate vasodilatators.

Animals↗

PubMed1996

K+ channels and the intracellular calcium signal in human melanoma cell proliferation.

K+ channels, membrane voltage, and intracellular free Ca2+ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K+ channel and a calcium-activated K+ channel. The inwardly rectifying K+ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K+ channel blockers quinidine, tetraethylammonium chloride and Ba2+ and elevated extracellular K+ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca2+ followed by a long-lasting Ca2+ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca2+, illustrating a transmembrane flux of Ca2+ following its electrochemical gradient. We conclude that K+ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca2+, a messenger in the mitogenic signal cascade of human melanoma cells.

Animals↗

PubMed1996

Activation of Cl- currents in cultured rat retinal pigment epithelial cells by intracellular applications of inositol-1,4,5-triphosphate: differences between rats with retinal dystrophy (RCS) and normal rats.

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl--currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 microM inositol-1, 4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl- currents, blockable by 1 mm 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). IP3 activated Cl- currents in the presence of a high concentration of the calcium chelator BAPTA (10 mM) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10(-5)M Ca2+ via the patch pipette also led to a transient activation of Cl- currents. When the cells were preincubated in a bath solution containing thapsigargin (1 microM) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca2+ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl--current activation induced by IP3 which could be reduced by nifedipine (1 microM). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3.

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

PubMed1996

Membrane voltage and whole-cell currents in cultured pericytes of control rats and rats with retinal dystrophy.

PURPOSE: Retinal vascular changes are associated with retinitis pigmentosa in man and the retinal dystrophy of the Royal College of Surgeons (RCS) rat. Recently we demonstrated that retinal capillary pericytes possess electrical membrane characteristics typical for smooth muscle cells and may thus regulate retinal blood flow in vivo. METHODS: In the present study we compared cultured pericytes of 5-7 day old RCS and control rats by measurement of membrane voltage (Vmem) with microelectrodes and currents with the whole-cell configuration of the patch-clamp technique. RESULTS: Resting membrane voltage (-37.6 mV +/- 1.0 mV, n = 106 and -36.6 mV +/- 0.7 mV, n = 102, respectively) and whole-cell currents, which are mainly determined by a potassium conductance, were comparable in pericytes of control and RCS rats. The electrogenic component of the Na(+)-K(+)-ATPase in RCS rat pericytes was reduced, because depolarization of their membrane voltage by Na(+)-K(+)-ATPase inhibitor ouabain (10(-4) mol.1-1 or K+ free solution was significantly decreased compared to control pericytes. Norepinephrine (10(-5) mol.1-1) depolarized Vmem of control rat pericytes by delta 7.0 mV +/- 1.3 mV (n = 7), whereas it hyperpolarized Vmem of RCS rat pericytes by delta 2.0 mV +/- 0.8 mV (n = 11). The depolarization of Vmem by histamine (10(-5) mol.1-1) was significantly reduced in pericytes of RCS rats (delta 2.6 mV +/- 0.4 mV, n = 7) compared to control pericytes (delta 4.3 mV +/- 0.5 mV, n = 6). In pericytes of RCS rats ATP-induced activation of inward and outward currents and depolarization of membrane voltage were also significantly reduced. CONCLUSIONS: The data indicate that there are differences in the electrophysiological properties of retinal capillary pericytes of 5-7 days old RCS and control rats. Whereas resting membrane voltage and K+ conductance are comparable in both groups, there are significant differences in the electrical activity of Na(+)-K(+)-ATPase and in the effects of vasoactive substances on the membrane voltage and currents. These differences might contribute to the vascular changes observed in RP, and possibly accelerate the progress of retinal dystrophy.

Adrenergic alpha-Agonists↗

PubMed1996

Contractile response of the isolated trabecular meshwork and ciliary muscle to cholinergic and adrenergic agents.

To characterize the contractile properties of isolated trabecular meshwork strips, we measured the effect of various cholinergic and adrenergic substances on the contractility of trabecular meshwork (TM) strips in comparison with the effects on ciliary muscle (CM) strips. Using an electromagnetic force length transducer we performed measurements of isometric tension on isolated bovine TM and CM strips. Isolated strips were contracted by cholinergic agonists, the relative potency being carbachol > pilocarpine > acetylcholine. The half-maximal effective concentration was 2 x 10(-7) mol l(-1) for carbachol and 2 x 10(-6) mol l(-1) for pilocarpine. To characterize muscarinic receptors, we tested selective antagonists for M1 (pirenzepine) and M3 (4-DAMP). Pharmacologically, the functional muscarinic receptors are of the M3 subtype in TM as well as CM. The M1 subtype seems to be less important. The alpha 1-agonist phenylephrine was more effective in inducing contractions in TM than in CM. The alpha 2-agonist brimonidine induced contractions only in TM. In precontracted tissues the beta-agonist isoproterenol induced a relaxation in both tissues. This relaxation could be inhibited by metipranolol. Epinephrine (or dipivefrin) induced small contractions in TM and CM, which became more prominent, especially in TM, when the beta-adrenoreceptors were inhibited by metipranolol. The data indicate the presence of functional muscarinic, alpha-adrenergic, and beta-adrenergic receptors in bovine TM and CM. The contractile properties of TM and CM are differently modulated by the various drugs. Cholinergic and alpha-adrenergic agonists induced contraction, whereas beta-agonists induced relaxation.

Adrenergic Agents↗

PubMed1995

Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye.

Contractile properties of isolated trabecular meshwork strips have recently been described. In the present paper we characterize the regulation of the outflow pathway in the isolated perfused anterior segment of the bovine eye. Anterior segments of bovine eyes with detached iris, ciliary body and ciliary muscle were perfused at constant pressure of 8.8 mmHg. A constant outflow of approximately 6-8 microliters min-1 could be obtained for at least 3 hr. The calculated outflow resistance was in the range 1.1-1.4 mmHg min microliter-1. The relative outflow was significantly reduced after application of carbachol, reaching a maximal inhibition of 30%. EC50 for carbachol was 3 x 10(-8) mol l-1. Atropin completely blocked the effect of carbachol on outflow. Morphological examination of perfused anterior segments which were perfused with carbachol revealed an intact fine structure of the meshwork cells. Pilocarpine at 10(-5) mol l-1 reduced outflow by 15%. Epinephrine at 10(-5) mol l-1 reduced outflow, while epinephrine at 10(-6) mol l-1 slightly increased the outflow rate. This effect could be blocked by metipranolol. Endothelin-1 in concentrations of 2 x 10(-9) and 2 x 10(-8) mol l-1 inhibited relative outflow by > 30%. Carbachol, pilocarpine, endothelin and a high dose of epinephrine, which have been shown to induce contractions in isolated bovine trabecular meshwork and ciliary muscle strips, induced a reduction of outflow rate and an increase of outflow resistance of the anterior segment. Thus, at least in the bovine eye, the trabecular meshwork per se is directly involved in the regulation of aqueous humor outflow.

Animals↗

PubMed1995

Incomplete belts of tight junctions in cultured non-pigmented human ciliary epithelial cells.

Tight junctions of cultured human non-pigmented ciliary epithelial cells were studied with the freeze-fracture technique and related to the transepithelial electrical resistance of these monolayers. Isolated tight junctional fibrils or small groups and networks of tight junctions sometimes associated with gap junctions were revealed in freeze-fracture images of the lateral plasma membrane. The tight junctions always formed incomplete belts, so that the apical and basolateral plasma membrane domains often were in continuity without morphological evidence of interposed intercellular junctions. The monolayers revealed a transepithelial resistance of 19.7 +/- 2.1 omega.cm2. Protamine induced a reversible increase of the transepithelial resistance of the cultures by 91 +/- 12%, but still the tight junctions formed incomplete belts. We conclude that contrary to complete networks of tight junctions in native non-pigmented ciliary epithelium, cultured monolayers only express incomplete belts of tight junctions which may be the morphological correlate of the relatively low transepithelial resistance of these monolayers. Interpretations on transepithelial transport and permeability characteristics of these cultures have to take into account the differences in junctional morphology from their native epithelium.

Cells, Cultured↗

PubMed1994

Electrophysiological properties of cultured human trabecular meshwork cells.

Previous studies using cultured bovine trabecular meshwork cells demonstrated at least two different cell types which were distinguishable morphologically and electrophysiologically. The purpose of the present study was to evaluate the electrical membrane properties of cultured human trabecular meshwork cells. Seven different human trabecular meshwork cell lines were grown from four different donors. One trabecular meshwork cell line was transformed by microinjection with SV40 DNA. The electrochemical properties of these TM cells were determined by micropuncture with glass microelectrodes. The mean membrane voltage at resting conditions differed between the cell lines (-33.3 to -58.7 mV). Application of 10-mmol l-1 Ba2+ induced repetitive voltage spikes in all cell lines. The voltage transients similar to action potentials were inhibited by nifedipine, but insensitive to tetrodotoxin. Acetylcholine evoked depolarizations in three cell lines which were blocked by atropine. In one cell line isoproterenol caused sustained depolarizations sensitive to metipranolol. All three of the cell lines tested depolarized upon application of the vasoactive peptide endothelin-1. All untransformed cell lines showed voltage spikes typical for smooth muscle cells and functional receptors for endothelin-1 and cholinergic agonists. One out of three cell lines tested possessed beta-adrenergic receptors influencing the membrane voltage.

Acetylcholine↗

PubMed1994

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, Ca(2+)-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 X 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 X 10(-5) mol/l) blocked from the intracellular side. Tetraethylammonium induced a dose-dependent block from the outside only with a half-maximal blocking concentration of 2.5 X 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 channel Po 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide.

PURPOSE: Recent evidence suggests that nitric oxide (NO) is a major messenger molecule regulating smooth muscle contractility. A role for NO in aqueous humor dynamics, and thus regulation of intraocular pressure, has been postulated. Recently, we described contractile properties of isolated bovine trabecular meshwork and ciliary muscle strips. To assess whether vasodilators contribute to the regulation of trabecular meshwork and ciliary muscle contractility, we measured the effect of various substances known to induce vasodilation by increasing intracellular cGMP production. METHODS: Measurements of isometric tension were performed on isolated bovine ciliary muscle and trabecular meshwork strips using a custom-built electromagnetic force-length transducer. The effects of a membrane-permeable cGMP and an inhibitor of nitric oxide formation (L-nitroarginine = L-NAG) were investigated. Organic nitrate (isosorbide dinitrate = ISDN, isosorbide-5-mononitrate = 5-ISMN) and non-nitrate (sodium nitroprusside = SNP, S-nitroso-N-acetyl penicillamine = SNAP) vasodilators were tested. RESULTS: Isolated strips were precontracted by carbachol 10(-6) mol/l for 30 minutes (100% carbachol maximal contraction). 8-bromo-cGMP 10(-4) mol/l evoked a relaxation to 86.7% +/- 1.4% (n = 8) in ciliary muscle and 58.6% +/- 5.4% (n = 7) in trabecular meshwork. Inhibition of NO-synthase by L-NAG increased the carbachol-induced contraction. The organic nitrovasodilators ISDN and 5-ISMN produced significant relaxations. The non-nitrates SNP and SNAP were the most potent relaxants. SNP 10(-4) mol/l relaxed the isolated ciliary muscle to 55.5% +/- 3.5% and the trabecular meshwork to 38.6% +/- 3.6%. ISDN and SNP were also tested on isolated strips without carbachol-induced precontraction. Both vasodilators had significant relaxing activity under these conditions. CONCLUSION: The data indicate that an increase of intracellular cGMP by application of cGMP and organic nitrate or non-nitrate vasodilators induces relaxation of the bovine trabecular meshwork and ciliary muscle. Thus, nitric oxide is a cotransmitter of smooth muscle relaxation in the chamber angle and may be involved in the regulation of aqueous humor dynamics.

Amino Acid Oxidoreductases↗

PubMed1993

Bovine and porcine large intestine as model epithelia in a student lab course.

A short-circuit current experiment on epithelial ion transport is described that is suitable for student classes in human and animal physiology. Segments of late distal colon from either pig or cow are obtained from the slaughterhouse depending on the animals' daily schedule. Initial tissue preparation already in the slaughterhouse, cold storage, and proper choice of bath solutions are essential prerequisites for success. Students monitor spontaneous transepithelial voltage and short-circuit current (Isc) by use of manually operated voltage clamp units. Two main transport mechanisms are studied, electrogenic Na+ absorption and Cl- secretion. Electrogenic Na+ absorption is studied by measuring the Isc drop after amiloride. Then Cl- secretion is stimulated by theophylline and subsequently inhibited by furosemide. In some experiments K+ secretion can be detected by the blocking effect of mucosal Ba2+. Response of tissues from pig and cow is qualitatively similar but quantitatively different. The equipment is sturdy and inexpensive, can be provided by most departmental workshops, and has been tested for 3 yr in regular lab courses. Observations made during these experiments are closely related to clinical states, such as secretory diarrhea, cystic fibrosis, and hyperaldosteronism, as well as to the mechanisms of clinically used diuretics.

Animals↗

PubMed1993

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 delta V = 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 (delta V = 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 (delta V = 9.6 +/- 2.0 mV, n = 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Insulin-induced hyperpolarization in retinal capillary pericytes.

PURPOSE: This study investigated the mechanism of insulin-induced membrane voltage hyperpolarization in retinal capillary pericytes, which possess electrical membrane properties typical for smooth muscle cells and are supposed to regulate retinal microcirculation by a contractile mechanism. METHODS: The mechanism of insulin-induced hyperpolarization was studied in cultured bovine retinal capillary pericytes using conventional microelectrodes. RESULTS: Resting voltage averaged -28 +/- 0.9 mV (mean +/- SEM, n = 45). Insulin (10(-9) to 10(-7) mol/l) induced a slow hyperpolarization in a dose-dependent fashion. Voltage change (delta V) was -3.1 +/- 0.4 mV (n = 14, P < 0.0001, = control) with an insulin concentration of 10(-8) mol/l. Blockade of potassium channels with Ba2+ (5 mmol/l) completely abolished the hyperpolarizing effect of insulin (n = 5). Apamin (10(-9) mol/l), a blocker of low-conductance Ca(2+)-activated potassium channels, also completely inhibited the insulin-induced hyperpolarization (n = 4). Blocking ATP-sensitive potassium channels with glibenclamide (10(-7) mol/l) did not reduce the hyperpolarizing action of insulin (delta V = -2.2 +/- 0.4 mV, n = 5, P = 0.29). Equivalent hyperpolarizations were recorded when insulin was added in the presence of ouabain (10(-4) mol/l) to inhibit the electrogenic Na+/-/K+/-ATPase (delta V = -3.5 +/- 1.0 mV, n = 4, P = 0.68). When pericytes were grown for 3 days in culture medium with elevated glucose concentrations (22.5 mmol/l), the resting membrane voltage and the insulin-induced hyperpolarization were not significantly altered. CONCLUSION: Insulin hyperpolarizes the membrane voltage of retinal pericytes probably mediated by activation of apamin-sensitive Ca(2+)-activated potassium channels. Therefore, hormonal modulation of membrane voltage by insulin might be an important factor in the regulation of pericyte contractility and retinal microcirculation under physiological conditions and in diabetes mellitus.

Adenosine Triphosphate↗

PubMed1992

Na(+)-dependent HCO3- transport and Na+/H+ exchange regulate pHi in human ciliary muscle cells.

We investigated intracellular pH (pHi) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4',5'-dimethylfluorescein (CDMF). The steady-state pHi was 7.09 +/- 0.04 (n = 12) in CO2/HCO3(-)-buffered and 6.86 +/- 0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11 +/- 0.06 pH units (n = 12) in the presence of CO2/HCO3- and by 0.91 +/- 0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pHi recovery, which was almost completely amiloride-sensitive in the absence of CO2/HCO3- but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25 +/- 0.02 (n = 4) in 6 min, while amiloride had no effect. The pHi recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pHi recovery was almost completely mediated by Na+/H+ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amiloride-insensitive pHi recovery was observed in CO2/HCO3(-)-buffered solution which was mediated by chloride-independent and chloride-dependent Na+ HCO3- cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter), was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pHi recovery after NH4Cl prepulse revealed Vmax = 0.57 pH units/min, Km = 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and Vmax = 0.19 pH units/min, Km = 14.3 mmol/liter extracellular sodium for the amiloride-insensitive component. We conclude that Na+/H+ exchange and chloride-independent and chloride-dependent Na(+)-HCO3- cotransport are involved in the pHi regulation of cultured human ciliary muscle cells.

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

PubMed1992

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 typical 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 mumol/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 mumol/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 La(3+)- and Ni(2+)-sensitive but verapamil-insensitive mechanism.

Acetylcholine↗

PubMed1992

Elevation of cytosolic free calcium in cultured ciliary epithelial cells by histamine: effects of verapamil and staurosporine.

Treatment with histamine (10(-4) M) of cultured non-pigmented human ciliary epithelial cells led to a biphasic elevation of free intracellular calcium mediated by H1-receptors. The initial transient increase was due to Ca(2+)-release from intracellular calcium stores and could be blocked with a high concentration of verapamil (10(-4) M). The subsequent sustained elevation of cytoplasmic calcium caused by an influx of extracellular calcium was reduced by staurosporine (10(-7) M). We conclude that the sustained increase of cytoplasmic calcium by histamine may be partially mediated by activation of protein kinase C. Since depolarization of the cells had no effect on intracellular calcium, we conclude that typical voltage-operated calcium channels do not significantly influence intracellular calcium.

Alkaloids↗