Biomedical subjects

J L Rae

Publications and source records attributed to J L Rae.

At least 19 recordsLinked to original sources

PubMed1992

Transient outwardly rectifying potassium channel in the rabbit corneal endothelium.

Ionic currents from freshly dissociated rabbit corneal endothelial cells were examined using patch-clamp technology and a perforated patch technique. Whole-cell current recordings revealed a transient outward K(+)-selective current that was blockable in a dose-dependent manner by 4-aminopyridine (4-AP) and quinidine. This current is similar to the 'A'-type current present in many excitable cells and is the first reported instance of such a current in any epithelial cell type. In addition to the transient current, an outwardly rectifying nonselective cation current was also observed. This current is also blocked by quinidine. To examine the possible role of these currents in the stromal volume regulatory function of the endothelium, corneas were perfused under a specular microscope with a glutathione-bicarbonate Ringer's solution (GBR) or GBR plus either 1 mM quinidine or 10 mM 4-AP. For quinidine perfusions, control corneas swelled at a rate of 6 microns/hr, while quinidine-perfused corneas swelled at a rate of 48 microns/hr. For 4-AP perfusions, control corneas swelled at a rate of -2 microns/hr, while 4-AP perfused corneas swelled at a rate of 24 microns/hr. One possible mechanism of the stromal swelling induced by these K+ channel blockers may be the result of loss of the K+ recycling pathway necessary for proper Na+/K+ ATPase function.

Animals↗

PubMed1992

Whole-cell potassium current in rabbit corneal epithelium activated by fenamates.

Rabbit corneal epithelium contains a large-conductance, potassium-selective channel, which is a major contributor to the whole-cell current. In perforated-patch recordings of the macroscopic current, the isolated cells studied had resting voltages of -41 +/- 20 mV and capacitances of 5.8 +/- 2.6 pF (mean +/- SD for n = 255). Activation of the channels was weakly voltage dependent. They opened at about -100 mV and reached an open probability of about 0.2 at +100 mV. The current was blocked by millimolar concentrations of external Ba2+ and quinidine. Diltiazem also blocked when applied to the external surface of the membrane. Nonsteroidal anti-inflammatory agents of the fenamate group were powerful activators of the channel at submillimolar concentrations when applied either to the inside or the outside of the channels. The mechanism of action which leads to his activation is not yet known.

Animals↗

PubMed1992

Regulation of a potassium-selective current in rabbit corneal epithelium by cyclic GMP, carbachol and diltiazem.

The effects of cyclic GMP (cGMP), carbachol and diltiazem on a potassium-selective, delayed-rectifier current in freshly dissociated rabbit corneal epithelial cells were studied using a modified perforated-patch-clamp technique. The current was stimulated by both 500 microM cGMP (2.3-4.5-fold, mean = 2.9) and 250 nM carbachol, a muscarinic agonist (1.12-7.04-fold, mean = 3.8), and the stimulated current was totally blocked by diltiazem (10 microM). The effects of cGMP appeared to be, at least in part, different from those of carbachol as they required the presence of external calcium. Single-channel data suggest that cGMP and carbachol activate the potassium current by increasing the open probability of the channel via a second-messenger system and that the action of diltiazem is probably through a direct blocking effect on the open channel.

Animals↗

PubMed1992

A method for exceptionally low noise single channel recordings.

We present a method whereby, with integrating electronics, quartz patch electrodes and a novel use of silicone oil, background noise levels as low as .083 pA RMS in a 5 kHz bandwidth (4-pole Butterworth filter) have been achieved in single channel patch clamp recordings. These approaches result in much higher signal to noise ratios for single channel recording than have previously been reported and should allow many investigators to significantly reduce noise at a constant bandwidth or to increase their recording bandwidths by several kHz.

Animals↗

PubMed1992

Dye coupling in the corneal endothelium: effects of ouabain and extracellular calcium removal.

The effects of ouabain and extracellular calcium removal on gap junctional coupling of isolated rabbit corneal endothelium was examined using a modified dye-spread technique. This technique is a modification of a microelectrode procedure that now utilizes patch electrodes connected to a current-clamp circuit for dye iontophoresis and a shuttering system in the excitation light path to reduce phototoxic effects in the monolayer. It was found that a significant degree of junctional uncoupling occurred after 45 min of exposure to ouabain, quantified as a reduction in the effective diffusion coefficient of injected Lucifer yellow CH: 1.74 x 10(-7) cm2/s (control) versus 0.43 x 10(-7) cm2/s (ouabain-treated). It was also determined that no gap junctional uncoupling occurs after extended exposure (up to 3.5 h) to a calcium-free extracellular environment.

Animals↗

PubMed1992

Divalent cation effects on lens conductance and stretch-activated cation channels.

In patch clamp studies of apical membrane from frog lens epithelium, the most frequently observed channel is 'stretch-activated', highly selective for cations over anions but showing little selectivity for Na+ vs. K+. In normal physiological saline, the open channel conductance is 25-30 pS and quite linear over +/- 100 mV. In the absence of extracellular divalent ions, the open channel conductance for inward current flow increases to about 50 pS at the normal lens resting voltage of -75 mV, whereas the conductance for outward current flow is unaffected. In the intact lens, removal of extracellular divalents causes the input conductance approximately to double and the intracellular voltage to depolarize from -74 to -58 mV. A variety of divalent ions block this change in whole lens conductance and voltage in the same order in which they block the 'stretch channels'. Single voltage-clamped epithelial cells also increase their conductance when Ca2+ is removed from their bathing medium. There are, therefore, some striking parallels between the open channel properties of the 'stretch-activated' cation channel and the response of the whole lens or single lens cells to removal of extracellular Ca2+. There are also inconsistencies. This channel is apparently not open in the normal resting lens so removal of extracellular Ca2+ must cause it to open if it is indeed responsible for the increase in lens conductance. However, we have not been able to demonstrate convincingly an increase in open probability at the single-channel level when external divalents are removed.

Animals↗

PubMed1992

Constructing a patch clamp setup.

It should be obvious that there are many ways to construct clamp set-ups that are either equivalent or sufficient for the experiments planned. The hardware and electronics can be obtained from several manufacturers, as can analysis software. What we have presented here are guidelines primarily meant to point a new experimenter in the right direction and, we hope, to guide more experienced investigators toward techniques that can improve the resolution of their measurements.

Artifacts↗

PubMed1992

Glass technology for patch clamp electrodes.

Based on all of the properties of glass described here, it is obvious that no one glass can be recommended for all purposes and for all cells. Borosilicate glasses like 7760, 7052, and 7040 are good general purpose glasses for both single-channel and whole-cell recordings. They are good initial choices but, of course, must be checked for each cell type for problems associated with leaching of blockers, etc., from the glass. Corning 8161 is the best glass studied to date with respect to electrical and thermal properties but must be checked carefully for leachable components. If perforated-patch whole-cell recordings are to be used, 8161, KG-12, or some other high lead, low melting point glass are probably the best choices.

Data Collection↗

PubMed1992

Initial characterization of whole-cell currents from freshly dissociated corneal keratocytes.

The perforated patch technique was utilized to obtain whole-cell currents from freshly dissociated rabbit corneal keratocytes. We describe and provide the initial characterization of two distinct whole cell currents in rabbit keratocytes: a K(+)-selective delayed rectifier and a voltage-sensitive, tetrodotoxin blockable Na+ current. The voltage-sensitive Na+ current is of sufficient magnitude to allow us to initiate action potentials when current-clamping the cells. This is the first detailed electrophysiological study of corneal keratocytes.

Action Potentials↗

PubMed1992

Whole-cell currents from noncultured human lens epithelium.

Perforated patch techniques were used to measure whole-cell ionic currents in freshly dissociated human lens epithelial cells that had not been subjected to culture media or serum. With a 150 mmol/l K+ internal solution, the cells had resting voltages of -27.4 +/- 4.7 mV (mean +/- standard deviation [SD]) and capacitances of 10.4 +/- 2.8 pF (mean +/- SD). The input resistance of the cells was 1.6 +/- 0.7 G omega (mean +/- SD) at large negative voltages. A delayed outwardly rectifying K+ current was found in most cells studied. Current magnitudes of 1-2 nA at +80 mV were common. The current had selectivities, activation time constants, deactivation time constants, open probability versus voltage relationships, and inactivations similar to those of the delayed rectifying K+ current found in many cell types and studied previously in cultured human lens epithelium. These results verify the existence, at high density, of these currents in noncultured human epithelial cells.

Aged↗

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

Sodium channels in ocular epithelia.

Voltage-gated, tetrodotoxin(TTX)-blockable sodium channels are found in most excitable cells and are the primary contributors to action potentials generated by many of these cells. To date, there has only been one report of a non-cultured vertebrate epithelial cell type containing TTX-blockable Na+ channels: rabbit non-pigmented ciliary body epithelial cells [Cilluffo MC et al. (1991) Invest Opthalmol Vis Sci 32: 1619-1629], and three reports of cultured epithelial cells containing TTX-blockable Na+ channels: rabbit non-pigmented and pigmented ciliary body epithelium [Ciluffo MC et al. (1991) Invest Opthalmol Vis Sci 32: 1619-1629; Fain GL, Farahbakhsh (1989) J Physiol (Lond) 417: 83-103] and human lens epithelium [Cooper K et al. (1990) J Membr Biol 117: 285-298]. We report here the presence of sodium currents in two different non-cultured, freshly dissociated transporting epithelial cell types: the rabbit corneal endothelium and the frog lens epithelium. We also report the occurrence of sodium currents in six additional cultured ocular epithelial cell types from three different species. These currents have a current/voltage (I/V) relationship consistent with traditional voltage-gated Na+ currents, are quinidine- and TTX-blockable (of the low-affinity TTX-sensitive type), and disappear following bath substitution of Na+ with Cs+ or K+.

Animals↗

PubMed1991

Cyclic GMP regulation of a voltage-activated K channel in dissociated enterocytes.

Enterocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from -23 to -39 mV and 29 to 13 M omega, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at -80 mV and subsequently stepped to a series of depolarizing voltages (from -70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above -20 mV. This current was sensitive to holding potential and was not activated when the cells were held at -40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the current-carrying ion. BaCl2 (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC50 for charybdotoxin was 50 nM. Cyclic GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above -40 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B↗

PubMed1991

Cell to cell communication and pH in the frog lens.

Fiber cells of the lens are electrically and diffusionally interconnected through extensive gap junctions. These junctions allow fluxes of small solutes to move between inner cells and peripheral cells, where the majority of transmembrane transport takes place. We describe here a method utilizing two intracellular microelectrodes to measure the cell to cell resistance between fiber cells at any given distance into the intact lens. We also use ion-sensitive microelectrodes to record intracellular pH at various depths in the intact lens. We find that gap junctions connecting inner fiber cells differ in pH sensitivity as well as normal coupling resistance from those connecting peripheral cells. The transition occurs in a zone between 500 and 650 microns into the lens. Fiber cells peripheral to this zone have a specific coupling resistance of 1.1 omega cm2, whereas those inside have a specific coupling resistance of 2.7 omega cm2. However, when the cytoplasm of fiber cells is acidified by bubbling with CO2, peripheral cells uncouple and the cell to cell resistance goes up more than 40-fold, whereas junctions inside this zone are essentially unaffected by changes in intracellular pH. In a normal frog lens, the intracellular pH in fiber cells near the lens surface is 7.02, a value significantly alkaline to electrochemical equilibrium. Our data suggest that Na/H exchange and perhaps other Na gradient-dependent mechanisms in the peripheral cells maintain this transmembrane gradient. Deep in the lens, the fiber cell cytoplasm is significantly more acidic (pHi 6.81) due to influx of hydrogen across the inner fiber cell membranes and production of H+ by the inner fiber cells. Because of the normally acid cytoplasm of interior fiber cells, their loss of gap junctional sensitivity to pH may be essential to lens survival.

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↗

PubMed1991

Resting voltage measurements of the rabbit corneal endothelium using patch-current clamp techniques.

The resting potential (Em) of freshly isolated rabbit corneal endothelium was measured at room temperature (22 degrees C) and at 34 degrees C. Due to the wide range of values reported in the literature and the difficulty in obtaining long-term measurements using microelectrodes in these cells, a current-clamp technique was employed using whole cell patch-clamp electrodes. The electrodes contained a K+ methanesulfonate-based intracellular solution, and a NaCl/HCO3- Ringer's solution was used extracellularly. Three preparations of endothelium were examined: single dissociated cells, the isolated monolayer (stripped from the stroma with Descemet's membrane), and the intact isolated cornea. The perforated-patch technique, with amphotericin B in the electrode, was also used with the intact-cornea preparation at 34 degrees C. The mean Em values for the combined preparations at 22 degrees C and 34 degrees C were -35.3 mV and -55.0 mV, respectively; those for the intact-cornea preparation were -34.4 mV and -61.6 mV (at 22 degrees C and 34 degrees C, respectively). The isolated monolayer preparation showed a small but significant depolarization at both temperatures. These results demonstrate temperature dependence for Em in the corneal endothelium and show that more extensively dissected preparations have similar although not identical Ems to those of the intact cornea.

Amphotericin B↗

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↗