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J L Rae

Publications and source records attributed to J L Rae.

At least 37 records · Page 2Linked to original sources
PubMed1995

Calcium entry in rabbit corneal epithelial cells: evidence for a nonvoltage dependent pathway.

We performed experiments to elucidate the calcium influx pathways in freshly dispersed rabbit corneal epithelial cells. Three possible pathways were considered: voltage-gated Ca++ channels, Na+/Ca++ exchange, and nonvoltage-dependent Ca(++)-permeable channels. Whole cell inward currents carrying either Ca++ or Ba++ were not detected using voltage clamp techniques. We also used imaging technology and the Ca(++)-sensitive ratiometric dye fura 2 to measure changes in intracellular Ca++ concentration ([Ca]i). Bath perfusion with NaCl Ringer's solution containing the calcium channel agonist Bay-K-8644 (1 microM), or Ni++ (40 microM), a blocker of many voltage-dependent calcium channels, did not affect [Ca++]i. Membrane depolarization with a KCl Ringer's bath solution resulted in a decrease in [Ca++]i. These results are inconsistent with the presence of voltage gated Ca++ channels. Nonvoltage gated Ca++ entry, on the other hand, would be reduced by membrane depolarization and enhanced by membrane hyperpolarization. Agents which hyperpolarize via stimulation of K+ current, such as flufenamic acid, resulted in an increase in ratio intensity. The cells were found to be permeable to Mn++ and bath perfusion with 5 mM Ni++ decreased [Ca++]i suggesting that the Ca++ conductance was blocked. These results are most consistent with a nonvoltage gated Ca++ influx pathway. Finally, replacing extracellular Na+ with Li+ resulted in an increase in [Ca++]i if the cells were first Na(+)-loaded using the Na+ ionophore monensin and ouabain, a Na(+)-K(+)-ATPase inhibitor. These results suggest that Na+/Ca++ exchange may also regulate [Ca++]i in this cell type.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Calcium currents in human and canine jejunal circular smooth muscle cells.

BACKGROUND & AIMS: Although calcium plays an essential role in intestinal smooth muscle contractile activity, calcium entry pathways in canine and human small intestine are largely unknown. The goal of this study was to characterize calcium channels, a potential entry pathway for calcium, in isolated circular smooth muscle cells of canine and human jejunum. METHODS: Single freshly dissociated human and canine jejunal circular smooth muscle cells were studied using single-channel and perforated whole-cell patch clamp recordings as well as fluorescence dual wavelength ratio imaging. RESULTS: An inward whole-cell current was identified that was carried by a 17 pS (80 mmol/L Ba2+) dihydropyridine-sensitive, barium-permeable channel. The current was potentiated by BayK 8644 (1 mumol/L; n = 3; 82% +/- 34%), acetylcholine (1 mumol/L; n = 8; 42% +/- 5%), and erythromycin (1 mumol/L; n = 9; 70% +/- 11%) and was completely blocked by nifedipine (1 mumol/L; n = 6) or diltiazem (200 mumol/L; n = 4). Application of BayK 8644 (1 mumol/L), acetylcholine (1 mumol/L), or erythromycin (1 mumol/L) to Fura-2-loaded smooth muscle cells bathed in Krebs' solution containing 2.54 mmol/L calcium increased intracellular calcium levels. CONCLUSIONS: A calcium entry pathway was identified in canine and human jejunal circular smooth muscle cells. The pathway was mediated by a dihydropyridine-sensitive calcium channel. The channel allowed the entry of significant amounts of calcium at physiological extracellular calcium concentration.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Effect of Prozac on whole cell ionic currents in lens and corneal epithelia.

Prozac (fluoxetine), a compound used therapeutically in humans to combat depression, has substantial effects on ionic conductances in rabbit corneal epithelial cells and in cultured human lens epithelium. In corneal epithelium, it reduces the current due to the large-conductance potassium channels that dominate this preparation. Its effects seem largely to decrease the open probability while leaving the single-channel current amplitude unaltered. In cultured human epithelium, currents from calcium-activated potassium channels and inward rectifiers are unaffected by Prozac. Delayed-rectifier potassium currents are reduced by Prozac in a complicated way that involves both gating and single-channel current amplitude. Fast tetrodotoxin-blockable sodium currents are also decreased by Prozac in this preparation. For all of these ion conductance effects, Prozac concentrations of 10(-5) to 10(-4) M are required. Whereas these levels are 10- to 100-fold higher than the plasma levels achieved in therapeutic use in humans, they are comparable to or less than levels needed for many other blockers of the ionic conductances studied here.

Animals↗
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↗
PubMed1994

Carbon monoxide stimulates a potassium-selective current in rabbit corneal epithelial cells.

The effects of CO on ion currents in freshly dispersed rabbit corneal epithelial cells were assessed using the perforated patch whole cell voltage-clamp technique. Bath perfusion with 1% CO resulted in a 84 +/- 18% (mean +/- SE, n = 14) increase in potassium current (IK) and a membrane hyperpolarization from -42 +/- 4 to -51 +/- 4 mV. The CO-stimulated current reversed at -64 +/- 7 mV [reverse potential (EK) = -87 mV]. The stimulated current was blocked by 1 mM quinidine or 1 mM diltiazem, agents that inhibit IK in rabbit corneal epithelial cells. Single potassium-channel currents measured in the cell-attached configuration showed that exogenous CO increased the steady-state open probability from 0.003 to 0.156 at a holding potential of -40 mV. CO did not affect open probability in excised patches. The single-channel conductance measured from -40 to +40 mV was unaffected. Intracellular guanosine 3',5'-cyclic monophosphate (cGMP) concentration measured with radioimmunoassay techniques was found to increase from 0.41 +/- 0.24 to 0.55 +/- 0.27 pmol/10(6) cells after the addition of 1% CO (P < 0.05). The data show that bath perfusion with exogenous CO activates IK and hyperpolarizes the resting membrane potential; the data also suggest that CO modulates intracellular cGMP concentration.

Animals↗
PubMed1994

Outwardly rectifying potassium currents in lens epithelial cell membranes.

Isolated epithelial cells from chick, pig, monkey, rabbit, bovine, and human lenses contain K+ channels that often turn on with a delay after a voltage step and have a larger macroscopic conductance for outward currents than for inward currents even with the same K+ concentration on both sides of the membrane. These outward rectifiers are quite diverse between different lens types and more than one kind can be present even within a single lens species. The channels differ substantially in the voltage dependence of their opening, their deactivation time constants, and the time course of their inactivation. Most produce currents of the delayed rectifier type but others show similarities to A-type currents. Because these different channels open at different voltages, inactivate to different degrees and represent different fractions of the total conductance from one lens cell to another, their contribution to the resting voltage is not the same in all cells investigated. These currents are the most frequently occurring in bovine, pig, monkey, and human lens epithelium and also occur commonly in chick lens epithelium. They occur less frequently in rodents.

Animals↗
PubMed1993

Ion channel involvement in the temperature-sensitive response of the rabbit corneal endothelial cell resting membrane potential.

Previous studies have shown that the resting potential (Em) of the corneal endothelium hyperpolarizes following an increase in temperature above 24 degrees C. Whole-cell studies using the perforated-patch technique were used to compare currents and Em values from isolated corneal endothelial cells at 24 and 32 degrees C. These studies revealed a small, outwardly rectifying, slowly activating, weakly voltage-dependent current with a reversal potential showing K+ selectivity (Erev = -80 mV). This current had features similar to the whole-cell current seen following addition of HCO3- to these cells. Em measurements found an average 24 mV hyperpolarization following temperature elevation in NaCl Ringer. Single channel studies found the only change in channel activity following an elevation in temperature to be an increase in the open probability (Po) of a K+ channel previously reported in this cell type to be activated by external anions. Po (-30 mV) at 24 and 32 degrees C equaled 0.003 and 0.06, respectively. Increases in Po were found at all voltages examined. This increased Po can account for the magnitude of the hyperpolarization seen in these cells following temperature elevation. Addition of HCO3- along with elevated temperature produced a synergistic effect on the increase in Po along with an increased hyperpolarization of the cell, pointing to separate mechanisms of activation from these two stimuli.

Animals↗
PubMed1993

Regulation of a voltage-dependent, calcium-activated K conductance by cyclic GMP in dissociated flounder enterocytes.

Enterocytes from the winter flounder (Pseudopleuronectes americanus) were isolated by collagenase digestion and maintained in flounder Ringer's solution. Whole cell currents were studied using the amphotericin-perforated whole-cell patch clamp technique. The mean resting membrane potential and capacitance values or dissociated cells were -45 +/- 7 mV and 5 +/- 0.4 pF, respectively. Enterocytes held at -20 mV and treated with 1 mumol.l-1 ionomycin exhibited outward currents when cells were stepped through a series of voltages from -60 to +110 mV. The reversal potential of this current in flounder Ringer's solution was -55 mV and the voltage at which half-maximal activation occurred was +20 mV. Voltage-dependent inhibition of outward current was observed at +60 mV and above. When cells were bathed in symmetric K Ringer's solution the reversal potential shifted to zero mV and no inhibition of current was observed at voltages between -60 and 140 mV. When the holding potential of the cell was changed from -20 to -80 mV and stepped from -60 to +110 mV, a second [previously characterized, O'Grady et al. (1991)] K current with delayed-rectifier properties was identified. This observation demonstrated that the delayed rectifier K channel and the Ca(2+)-activated K channel described in this study exist in the same cell. Extracellular addition of 2 mmol.l-1 Ba2+ to cells bathed in symmetric K Ringer's solution resulted in nearly complete inhibition of outward current. Charybdotoxin produced only minor effects on this current.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

The use of quartz patch pipettes for low noise single channel recording.

Quartz has a dissipation factor of approximately 10(-4), which is an order of magnitude less than that of the best glasses previously used to fabricate patch pipettes; it's dielectric constant of 3.8 is also lower than that of other glasses. On the basis of these electrical characteristics it is expected that patch pipettes pulled from quartz tubing will produce significantly less noise than pipettes made from other glasses. Our work confirms these expectations and we describe theoretical and practical aspects of the use of quartz pipettes for single channel patch voltage clamp measurements. Methods for pulling quartz pipettes with a laser-based puller and coating them with low-loss elastomers are discussed, as are precautions that are necessary to achieve low noise recordings. We have shown that quartz pipettes can be pulled from tubing with outer diameter to inner diameter ratios as large as 3 and a method of applying heavy elastomer coatings all the way to the tip of pipettes is presented. Noise sources arising from the pipette and its holder are described theoretically, and it is shown that measured noise is in good agreement with such predictions. With low noise capacitive feedback electronics, small geometry holders, and thick-walled quartz pipettes coated with low-loss elastomers we have been routinely able to achieve noise of 100 fA rms or less in a 5-kHz bandwidth with real cell patches and a pipette immersion depth of approximately 2 mm. On occasion we have achieved noise as low as 60 fA rms in this bandwidth.

Animals↗
PubMed1993

Characterization of an outward potassium current in canine jejunal circular smooth muscle and its activation by fenamates.

1. A perforated patch clamp technique was used to study an outward potassium current in freshly dissociated circular smooth muscle cells of the canine jejunum. 2. A voltage-dependent outward current was identified which was highly potassium selective, weakly holding voltage sensitive, increased its open probability at -65 mV, and reached unit open probability at +5 mV. 3. Quinidine (0.1-1 mM) and tetraethylammonium ion (TEA) (10-50 mM), blocked the potassium current in a dose-dependent manner. Blockade of the outward potassium current was accompanied by membrane depolarization which reversed on removal of the blocker from the bathing solution. 4. Mefenamic and flufenamic acids, non-steroidal anti-inflammatory agents in the fenamate group, were potent activators of the current. Activation was accompanied by hyperpolarization of the membrane with a mean shift in the membrane voltage of 22 mV. 5. It was concluded that the outward potassium current is a major regulator of the resting membrane voltage in isolated circular smooth muscle cells of the canine jejunum. Fenamates activated this current with potentially profound effects on cellular excitability.

Animals↗
PubMed1993

Activation of whole cell currents in isolated human jejunal circular smooth muscle cells by carbon monoxide.

Carbon monoxide (CO) is a low molecular weight oxide produced endogenously from fatty acids and heme protein. A physiological role for CO has been suggested for vascular smooth muscle, hemostasis, and olfactory neurons, but direct evidence is lacking. Heme oxygenase, which catalyzes the formation of CO from heme proteins, is present in small intestinal smooth muscle. The effect of 1% CO on whole cell currents in normal human jejunal circular muscle cells was studied with the use of a perforated patch-clamp technique. A 1% CO-containing Krebs solution caused an initial and transient increase in whole cell current in 20 of 22 cells tested (175 +/- 40%, mean +/- SE) and a transient hyperpolarization (15.6 +/- 3.6 mV, mean +/- SE) of the membrane potential. During prolonged recordings, 1% CO evoked ongoing cyclic increases and decreases in the whole cell current. Each current increase was accompanied by a sharp membrane hyperpolarization. These data suggest that CO may modulate whole cell potassium current and membrane potential.

Carbon Monoxide↗
PubMed1993

Potassium current in circular smooth muscle of human jejunum activated by fenamates.

Thirty-eight cells, freshly isolated from circular smooth muscle of normal human jejunum obtained from nine patients undergoing weight-reduction surgery for morbid obesity, were patch clamped using a perforated patch-clamp technique. A highly potassium-selective voltage-dependent outward current was present in all cells. The current was carried by a 220-pS channel that activated near -75 mV and reached unit open probability at about +10 mV. Blockade of the current by quinidine (50 microM) and tetraethylammonium (25 mM) was accompanied by membrane depolarization to 0 to -3 mV, suggesting that this current was the major determinant of the membrane potential. Flufenamic and mefenamic acid at concentrations comparable with blood levels reached when these drugs are used in clinical therapy as nonsteroidal anti-inflammatory agents, activated the potassium outward current and hyperpolarized the membrane potential. The shift in the membrane potential for 250 microM flufenamic acid was -36 +/- 24 (SD) mV. Activation was rapid (seconds) and reversible. It was concluded that normal human jejunal circular smooth muscle cells have a highly potassium-selective outward current, which is the major determinant of the membrane potential and which is activated by fenamates.

4-Aminopyridine↗
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↗