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Biomedical subjects

J Dewey

Publications and source records attributed to J Dewey.

At least 19 recordsLinked to original sources
PubMed1995

A non-enzymatic method for lens decapsulation which leaves the epithelial cells attached to the fibers.

We have developed a method to mechanically decapsulate the lens yet leave the epithelial cells attached to the fibers. This method uses divalent ion chelaters to loosen the capsule-epithelial interactions and bumetanide to control cell swelling. Light microscopy, scanning electron microscopy, and in-vitro fluorescence microscopy demonstrate that the capsule is removed and the epithelial cells remain adherent to the fibers when these procedures are used. Ion channel activity and epithelial gap junction communication remain following decapsulation. This decapsulated lens preparation should prove useful for many kinds of lens studies.

Animals↗
PubMed1992

The large-conductance potassium ion channel of rabbit corneal epithelium is blocked by quinidine.

Basal layers of the rabbit corneal epithelium contain a large-conductance potassium ion (K+) selective channel (160-170 pS in 150 mM KCl). This channel previously was shown to be blocked by cesium and barium ions applied to its outer surface. By direct patch clamp experiments, it is shown that the channel also is blocked by quinidine in the 0.1-1 mM range when applied either to the outside or inside of membrane patches containing these channels. This additional pharmacologic tool should aid in identifying the individual currents that compose the macroscopic currents from corneal epithelial cells and eventually should help to provide a detailed assessment of their function.

Animals↗
PubMed1991

Inwardly rectifying potassium current in mammalian lens epithelial cells.

Lens potassium conductance is essential for the maintenance of lens volume and transparency. Recent work has identified three major potassium currents in lens: 1) an outwardly rectifying current, 2) an inwardly rectifying current, and 3) a calcium-activated current. This paper presents a study of the lens inward rectifier using whole cell and single-channel patch-clamp techniques. Inwardly rectifying potassium current is present in isolated human, rabbit, rat, and mouse lens epithelia. The voltage about which rectification occurs depends on the external potassium concentration. Internal magnesium is not necessary for rectification. In physiological saline, a time-dependent decrease in current during sustained hyperpolarization is seen. This "droop" is due to voltage-dependent block by external sodium. The inward rectifier is also effectively blocked by external cesium or barium but not by tetraethylammonium or 4-aminopyridine. The mouse lens inward rectifier has a single-channel conductance of 32 pS (measured on-cell with 150 mM potassium in the pipette). The single-channel current-voltage relationship is linear in the inward direction. In contrast to the macroscopic case, no outward current was measurable. The inward rectifier in lens has the necessary properties to be involved in setting resting voltage.

Animals↗
PubMed1990

Effects of indapamide on endothelium-dependent relaxations in isolated canine femoral arteries.

Indapamide is an effective antihypertensive agent in humans and in experimental hypertensive animals. The aim of the present study was to investigate whether indapamide affects endothelium-dependent and independent relaxations in canine femoral arteries. Rings (with or without endothelium) were contracted with prostaglandin F2 alpha (2 X 10(-6) mol/liter) before the addition, in a cumulative fashion, of relaxing agents. Indapamide (10(-7) to 10(-4) mol/liter) had no direct effect on unstimulated or prostaglandin-stimulated preparations; it did not alter relaxations of preparations with endothelium induced by acetylcholine, bradykinin, adenosine diphosphate or the calcium ionophore A23187. Similarly, it did not affect relaxations induced by sodium nitroprusside, prostacyclin or forskolin in preparations with or without endothelium. Indomethacin shifted the concentration-response curve to bradykinin to the right and did not alter that to the other relaxing drugs. The reduced relaxation to bradykinin was reversed in a concentration-dependent manner by indapamide (10(-7) to 10(-5) mol/liter). In the presence of indomethacin, indapamide shifted the concentration response curve to prostacyclin (in rings with endothelium) and to forskolin (in rings with and without endothelium) to the left. Thus, indapamide does not directly affect endothelium-dependent and independent relaxations. However, when prostanoid production is impaired, indapamide facilitates the release of endothelium-derived relaxing factor(s), and to a lesser extent, the direct action on vascular smooth muscle of prostanoids (prostacyclin) released from the endothelium.

Acetylcholine↗
PubMed1990

Electrophysiology of cultured human lens epithelial cells.

The lens epithelial K+ conductance plays a key role in maintaining the lens ionic steady state. The specific channels responsible for this conductance are unknown. We used cultured lens epithelia and patch-clamp technology to address this problem. Human lens epithelial explants were cultured and after 1-4 passages were dissociated and used in this study. The cells from which we measured had a mean diameter of 31 +/- 1 microns (SEM, n = 26). The resting voltage was -19 +/- 4 mV (SEM, n = 10) and the input resistance was 2.5 +/- 0.5 G omega (SEM, n = 17) at -60 mV. Two currents were prominent in whole-cell recordings. An outwardly rectifying current was seen in nearly every cell. The magnitude of this current was a function of K+ concentration and was blocked by 3 mM tetraethylammonium. The instantaneous current-voltage relationship was linear in symmetric K+, implying that the outward rectification was due to gating. The current showed complex activation and inactivation kinetics. The second current seen was a transient inward current. This current had kinetics very similar to the traditional Na+ current of excitable cells and was blocked by 0.1 microM tetrodotoxin. In single-channel recordings, a 150-pS K+ channel and a 35-pS nonselective cation channel were seen but neither account for the macroscopic currents measured.

Adult↗
PubMed1990

Potassium channel in rabbit corneal endothelium activated by external anions.

The apical membrane of the rabbit corneal endothelium contains a potassium-selective ionic channel. In patch-clamp recordings, the probability of finding the channel in the open state (Po) depends on the presence of either HCO3- or Cl- in the bathing medium. In a methane sulfonate-containing bath, Po is less than 0.05 at all physiologically relevant transmembrane voltages. With 0 mM [HCO3-]o at +60 mV, Po was 0.085 and increased to 0.40 when [HCO3-]o was 15 mM. With 4 mM [Cl-]o at +60 mV, Po was 0.083 and with 150 mM Cl-, Po increased to 0.36. Low Po's are also found when propionate, sulphate, bromide, and nitrate are the primary bath anions. The mechanism of action of the anion-stimulated K+ channel gating is not yet known, but a direct action of pH seems unlikely. The alkalinization of cytoplasm associated with the addition of 10 mM (NH4)2SO4 to the bath and the acidification accompanying its removal do not result in channel activation nor does the use of Nigericin to equilibrate intracellular pH with that of the bath over the pH range of 6.8 to 7.8. Channel gating also is not affected by bathing the internal surface of the patch with cAMP, cGMP, GTP-gamma-s, Mg2+ or ATP. Blockers of Na/H+ exchange, Na(+)-HCO3- cotransport, Na(+)-K+ ATPase and carbonic anhydrase do not block the HCO3- stimulation of Po. Several of the properties of the channel could explain some of the previously reported voltage changes that occur in corneal endothelial cells stimulated by extracellular anions.

Animals↗
PubMed1990

A non-selective cation channel in rabbit corneal endothelium activated by internal calcium and inhibited by internal ATP.

The apical membrane of the rabbit corneal endothelium contains a cation selective channel of 21.7 +/- 0.4 pS conductance which is activated by internal Ca2+ and inhibited by internal ATP. Patch clamp studies show the channel to be quite selective for cations over anions but not to select between Na+ and K+. In excised patches in the inside-out configuration, the open probability increases when the internal Ca2+ is raised above 10(-4) M. ATP decreases the open probability when its internal concentration exceeds 10 microM. At 1 mM ATP, no channel openings are ever seen. AMP and ADP also inhibit at least as well as does ATP. One microM AMP-PNP, a non-hydrolyzable ATP, also inhibits gating and so phosphorylation by a PO4(-3) cleaved from ATP is not the mechanism of ATP inhibition. The channel is apparently impermeable to divalent ions since neither Ba2+ nor Ca2+ carry any detectable current. Quinidine produces a flickery, weakly voltage dependent blockade when applied to the channel's cytoplasmic surface. Both Ca2+ and ATP work at concentrations not expected to occur inside healthy cells. It is presently uncertain if this is a physiologically important channel in these cells.

Adenosine Diphosphate↗
PubMed1990

Comparative induction of gene mutations and chromosome damage by 1-methoxy-1,3,5-cycloheptatriene (MCHT), 1. Results from a battery of standard tests.

The ability of 1-methoxy-1,3,5-cycloheptatriene (MCHT) to induce gene mutations and chromosome breaks has been examined in a battery of standard assays. MCHT was not mutagenic to 5 strains of Salmonella, with or without S9 fraction. In L5178Y TK+/- mouse lymphoma cells, MCHT induced TK-/- mutants in the presence but not in the absence of S9 fraction. In V79 Chinese hamster cells, MCHT induced azaguanine-resistant mutants in the presence and absence of S9 but the effect was considerably reduced in the absence of S9. MCHT resulted in no increases in chromosome aberrations in cultured human lymphocytes, with or without S9 fraction, neither was there any increase in micro-nucleated polychromatic erythrocytes in treated mice. MCHT thus appears on the basis of these results, to be possibly a specific gene mutagen (rather than clastogen) for mammalian cells. This uncommon mutagenicity profile has been investigated further in an accompanying paper (Cole et al., 1990) and has proved to be an oversimplification.

Animals↗
PubMed1990

A maxi calcium-activated potassium channel from chick lens epithelium.

The apical membrane of embryonic chick lens epithelium contains at high density, a large conductance K+ channel whose open probability is increased by Ca++ at the inner surface of the membrane and by depolarization. The conductance of the channel when it is fully open in symmetrical 150 mM K+ solutions is 214 +/- 3 pS (mean +/- std. error). The current through the channel is a function of the K+ concentration. Gating (open probability) at positive transmembrane voltages increases as the internal [Ca++] is raised above 10(-7) M. The open probability decreases monotonically as the transmembrane voltage is made more negative. The channel is at least 87 times more permeable to K+ than to Na+ or Li+ and shows appreciable permeability to Rb+ and NH4+. It has at least three subconductance levels amounting to approximately 3/4, 1/2, and 1/4 the fully open unitary conductance. The occurrence of these subconductance levels is highly variable from one patch to another. The channel is blocked by physiological levels of internal Na+ but not over a physiological voltage range. This block is partially overcome by elevated external K+. This K+ channel from chick lens epithelium is blocked by a number of compounds known to block BK channels in other tissues. Here we show that decamethonium and Ba++ are effective blockers when added to the inner bathing solution at concentrations greater than .1 mM. Tetraethylammonium, Cs+, quinine, quinidine and Ba++ are all effective blockers when applied to the outer side of the channel in the .1 mM - 5 mM range. With the exception of internal Ba++, all of these compounds produce a fast flicker-type blockade. We use a one-site model to quantify the blockade caused by these flicker producing agents. The voltage dependence of the blockade by Cs+ suggests that this channel probably allows multiple occupancy.

Animals↗
PubMed1990

Single potassium channels in corneal epithelium.

The basal cell layers of the rabbit and human corneal epithelia contain a frequently occurring ionic channel whose unitary currents can be recorded in cell-attached or excised membrane patches by use of a patch voltage clamp. The channel is highly conductive (165 pS in 150 mM K+ salts) and is very selective for K+ over Na+ (PK/PNa greater than 40:1). Its open probability is increased by the application of suction to the recording pipette although its gating is less sensitive to suction than that of many other "stretch-activated" channels reported. The current through the channel is a saturating function of the K+ concentration in the bathing solutions with half saturation occurring at 480 mM and a single-channel current at saturation (imax) of 31 pA. In the absence of applied suction, the open probability is extremely variable from patch to patch and shows little voltage dependence over the physiologic voltage range. The channel also gates frequently to several subconductance levels. It is blocked by external Cs+ and Ba+2 in the 0.1-10 mM range but not by most other K+ channel blockers. It is also partially blocked by Ca+2 at both its internal and external surfaces. Because of its novel properties (stretch activation and large conductance), it can be used to measure the input resistance and total capacitance of single dissociated cells.

Animals↗
PubMed1989

Properties of single potassium-selective ionic channels from the apical membrane of rabbit corneal endothelium.

The corneal endothelium from several species contains a highly conductive, flickery potassium selective channel in the apical membrane. The channel flicker is not due to blockade. The single channel has an inwardly rectifying current-voltage relationship under symmetrical ionic conditions. Based on reversal potential measurements, it is more than 40:1 selective for potassium over sodium. It also shows subconductance levels. In bathing solutions lacking chloride and bicarbonate, its open probability is less than 0.1 and is quite independent of voltage in the physiological voltage range. Bicarbonate in the bath or DIDs in the pipette increase channel activity. The channel is blocked by external cesium in the 0.5 to 5 mM range. The steepness of the voltage dependence of this blockade is consistent with multiple occupancy.

Animals↗