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Biomedical subjects

J L Rae

Publications and source records attributed to J L Rae.

At least 73 records · Page 4Linked to original sources
PubMed1989

Membrane and junctional properties of dissociated frog lens epithelial cells.

Individual cells and cell pairs were isolated from frog lens epithelium. Individual cells were whole cell voltage clamped and the current-voltage relationship was determined. The cells had a mean resting voltage of -54.3 mV and a mean input resistance of 1.4 G omega. The current-voltage relationship was linear near the cell resting voltage, but showed decreased resistance with large depolarization or hyperpolarization. Junctional currents between pairs of cells were recorded using the dual whole cell voltage-clamp technique. The corrected junctional resistance was 15.5 M omega (64.5 nS). The junctional current-voltage relationship was linear. A combination of ATP and cAMP, in the electrodes, stabilized junctional resistance. Currents recorded when uncoupling was nearly complete, showed evidence of single connexion gating events. A single-channel conductance of about 100 pS was prominent. Dye spread between isolated cell pairs was demonstrated using Lucifer Yellow CH in a whole cell configuration. Photodamage to the cells due to the dye was apparent. Dye loaded cells, in the presence of exciting light, showed decreased resting voltages, decreased input resistances and morphological changes. Glutathione (20 mM) delayed this damage.

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.

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PubMed1989

Dye and electrical coupling between cells of the rabbit corneal endothelium.

The corneal endothelium is known to contain gap junctions on its lateral borders. Here we show that the endothelial cells of the rabbit cornea are highly dye coupled. Lucifer Yellow CH introduced into a single cell through either an intracellular glass microelectrode or a patch electrode rapidly spreads into its neighboring cells at room temperature. We have used the analysis of Safranyos and Caveney (JCB 100:736-747, 1985) to place quantitative lower bounds on the effective diffusion coefficient of Lucifer Yellow CH iontophoresed into rabbit corneal endothelial cells. By this analysis, we obtain a value of 5.0 x 10(-8) cm2/sec. We also demonstrate that both dye and electrical coupling exists in cell pairs or small clusters of cells enzymatically dissociated from Descemet's membrane.

Animals↗
PubMed1989

The use of indium-111 labeled platelet scanning for the detection of asymptomatic deep venous thrombosis in a high risk population.

Five hundred indium-111 labeled platelet imaging studies (387 donor and 113 autologous) were performed postoperatively in 473 patients who had undergone total hip replacement, total knee replacement, or internal fixation of a hip fracture to detect occult deep venous thrombosis. All patients had been anticoagulated prophylactically with aspirin, warfarin sodium (Coumadin), or dextran. Thirty-four possible cases of proximal deep venous thrombosis were identified in 28 asymptomatic patients. To verify the scan results, 31 venograms were performed in 25 patients (three refused). In 21 of 31 cases, totally occlusive thrombi were detected; in 5 cases, partially occlusive thrombi were detected; in 5 cases, no thrombus was seen. No patient who had a negative scan nor any patient who had a verified positive scan (and received appropriate heparin therapy) subsequently developed symptoms or signs of pulmonary embolism. One hundred forty-one indium study patients also underwent Doppler ultrasonography/impedance plethysmography (Doppler/IPG) as a comparative non-invasive technique. In 137 cases, the results of the indium study and Doppler/IPG studies were congruent. The indium study had no false negative results that were detected by Doppler/IPG. No patient had any clinically evident toxicity. These results suggest that indium-111 labeled platelet scanning is a safe, noninvasive means for identifying DVT in high risk patients.

Adult↗
PubMed1989

Potassium channels in chick lens epithelium change with maturation.

Before hatching, chick embryos show a high incidence of inwardly rectifying potassium channels and calcium-activated potassium channels in the apical membrane of their lens epithelium. Subsequent to hatching, these channel types disappear while, at about the same time, a new calcium insensitive maxi potassium channel appears. This new channel shows several properties in common with the calcium-activated maxi potassium channels but is different in its conductive and gating properties.

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PubMed1988

Pharmacologic blockade of ionic channels in lens membranes.

Channel blockers are compounds that disrupt the flow of current through ionic channels in membranes. We use the patch clamp technique to characterize the interactions of blocking particles with individual lens membrane channels. The mean current amplitude at many voltages is determined. The ratios of blocked to unblocked currents at several blocker concentrations are compared to a one binding site model to determine the kd and electrical distance of the site. When channel flickers are too fast to be resolved, the calculation of the power spectral density of the open channel current is useful. It is suggested that channel blockers might be useful therapeutically in some types of cataracts.

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PubMed1986

A cation channel in frog lens epithelia responsive to pressure and calcium.

Patch-clamp recording from the apical surface of the epithelium of frog lens reveals a cation-selective channel after pressure (about +/- 30 mm Hg) is applied to the pipette. The open state of this channel has a conductance of some 50 pS near the resting potential (-56.1 +/- 2.3 mV) when 107 mM NaCl and 10 HEPES (pH 7.3) is outside the channel. The probability of the channel being open depends strongly on pressure but the current-voltage relation of the open state does not. With minimal Ca2+ (55 +/- 2 microM) outside the channel, the current-voltage relation is nonlinear even in symmetrical salt solutions, allowing more current to flow into the cell than out. The channel, in minimal Ca2+ solution, is selective among the monovalent cations in the following sequence K+ greater than Rb+ greater than Cs+ greater than Na+ greater than Li+. The conductance depends monotonically on the mole fraction of K+ when the other ion present is Li+ or Na+. The single-channel current is a saturating function of [K+] when K+ is the permeant ion, for [K+] less than or equal to 214 mM. When [Ca2+] = 2 mM, the current-voltage relation is linearized and the channel cannot distinguish Na+ and K+.

Animals↗
PubMed1986

Potassium channels from chick lens epithelium.

The technique of patch-voltage clamp has been used to demonstrate several different kinds of K+ channels in the apical membrane of the chick lens epithelium. These include a 200- to 250-ps Ca2+-activated channel, a 200- to 250-ps non-Ca2+-activated channel whose probability of being open increases with hyperpolarization, a 35-ps flickery channel, and a 30-ps inward rectifier, all characterized in symmetrical 150 mM K+. The inward rectifier allows little if any outward current. The probability that the channel is open increases as the membrane patch is depolarized, whereas the mean open and closed times of the channel decrease with depolarization. It is proposed that the rectification is a property of the open channel rather than of its gating. External Cs+ at micromolar concentrations produces a flickery block that increases with hyperpolarization and blocker concentration. The mean open time inside bursts decreases with increasing blocker concentration, whereas the mean intraburst closed time is unaffected. Thus, Cs+ blocks the open channel. The steepness of the voltage dependence of the block suggests that multiple occupancy of the channel is possible.

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PubMed1985

Cell-to-cell fusion of lens fiber cells in situ: correlative light, scanning electron microscopic, and freeze-fracture studies.

We have discovered cell-to-cell fusion between fiber cells of adult frog lenses in situ. Stereo scanning electron microscopy (SEM) revealed fusion between neighboring fiber cells in radial cell columns (RCCs) and in the same growth ring, respectively. Cell-to-cell fusion of fiber cells in the lens produced fusion zones that in cross-section were larger and of different polygonal shapes than unfused fiber cells. The shape and sizes of fiber cells surrounding fusion zones and the alignment of RCCs were also altered. Serial sectioning through fusion zones confirmed that they were areas of cell-to-cell continuity established by the union of neighboring fiber cells as seen by SEM. Fusion zones represent a previously unrecognized intercellular pathway in the adult frog lens. Although numerous fusion zones were seen throughout the lens cortex and nucleus, cell-to-cell fusion was rarely observed to have occurred between elongating fiber cells. Interestingly, communicating junctions with an unusual ultrastructure that closely resembles the appearance of membranes in the process of fusion demonstrated in other systems were frequently seen in the region of the superficial cortex where fusion zones were most numerous. The fact that such unusual communicating junctions were not found in any other region of the lens leads us to speculate that structural changes in fiber cell communicating junctions may herald the formation of fusion zones and that the initial site of cell-to-cell fusion between fiber cells may be within communicating junctional plaques.

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PubMed1985

The localization of transport properties in the frog lens.

The selectivity of fiber-cell membranes and surface-cell membranes in the frog lens is examined using a combination of ion substitutions and impedance studies. We replace bath sodium and chloride, one at a time, with less permeant substitute ions and we increase bath potassium at the expense of sodium. We then record the time course and steady-state value of the intracellular potential. Once a new steady state has been reached, we perform a small signal-frequency-domain impedance study. The impedance study allows us to separately determine the values of inner fiber-cell membrane conductance and surface-cell membrane conductance. If a membrane is permeable to a particular ion, we presume that the conductance of that membrane will change with the concentration of the permeant ion. Thus, the impedance studies allow us to localize the site of permeability to inner or surface membranes. Similarly, the time course of the change in intracellular potential will be rapid if surface membranes are the site of permeation whereas it will be slow if the new solution has to diffuse into the intercellular space to cause voltage changes. Lastly, the value of steady-state voltage change provides an estimate of the lens' permeability, at least for chloride and potassium. The results for sodium are complex and not well understood. From the above studies we conclude: (a) surface membranes are dominated by potassium permeability; (b) inner fiber-cell membranes are permeable to sodium and chloride, in approximately equal amounts; and (c) inner fiber-cell membranes have a rather small permeability to potassium.

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PubMed1985

Transport properties of the lens.

Many studies have shown that the lens is a multicellular syncytial tissue whose electrophysiological properties are the integrated result of membrane transport, low-resistance gap junctions interconnecting the cells, and the restricted extracellular space between cells. There are at least three structurally distinct populations of cells within the lens, and the membrane transport properties of each cell type appear to differ. Indeed, there may be subcellular specialization of membrane transport properties in the surface epithelial cells. We review the physical structure of the lens, its electrical structure, and our present knowledge of the membrane transport properties of the different cell types. Our recent work has focused on radially circulating fluxes generated by the spatial localization of membrane transport in surface cell membranes versus inner fiber cell membranes. We review this work and present some simplified models of the results with some discussion of physiological implications.

Animals↗
PubMed1985

A correlative freeze-etch and electrophysiological study of communicating junctions in crystalline lenses.

We have conducted a correlative electrophysiological and morphological study of cell to cell coupling in frog and rat lenses. Electrical impedance measurements from frog and rat lenses were curve fit to a model of lens structure to obtain a value for internal resistivity (Ri). The mean and standard deviation of Ri was 550 +/- 190 ohm-cm (n = 7) in rat lenses and 3400 +/- 340 ohm-cm (n = 10) in frog lenses. These results indicate that the extent of cell to cell coupling is far more extensive in rat lenses than in frog lenses and therefore suggest that rat lens fiber cells are conjoined by greater numbers of communicating junctions than frog lens fiber cells. Freeze-etch replicas were made of fiber cells from rat and frog lenses of comparable size and from a comparable area (intermediate cortex) as that used in the electrophysiological study. A total of 987 and 1,393 square microns of replicated membrane were examined in rat and frog lenses, respectively. 1,573 communicating junctions were counted in rat lens replicas cumulatively measuring 313 square microns or 31.7% of the total membrane area. 604 communicating junctions were counted in frog lens replicas cumulatively measuring 163 square microns or 11.7% of the total membrane area. These results demonstrate that the amount of communicating junction predicted to be necessary to account for the more extensive electrotonic coupling between fiber cells in rat lenses than in frog lenses is qualitatively confirmed by morphological analysis.

Animals↗
PubMed1985

The application of patch clamp methods to ocular epithelia.

The techniques of patch voltage clamping and whole cell clamping have been applied to the lenses and corneas of several species of animals. Numerous ion channels have been found in the basal and apical membranes of lens epithelial cells, anterior and posterior surface lens fibers, apical membrane of corneal endothelial cells, and apical membrane of the second layer of corneal epithelial cells. No ion channels have been found in deep lens fiber membranes to date. There are 9-11 different kinds of potassium channels in ocular epithelial membranes, several different kinds of non-selective cation channels, and one non-selective channel with a large unit conductance. Sodium selective channels are seen only rarely while chloride selective channels have not been seen at all. Several channels have not yet been identified unequivocally. Using the gigohm seal technique, it is possible to show that the frog lens epithelial cell membrane is dominated by potassium channels. Also, a technique is described for using the reversal potential of a 25-30 pS non-selective cation channel to measure the resting voltage of epithelial cells without penetrating them. The results of lens ion channel localization studies are in only qualitative agreement with previous lens channel localization studies which used whole lens impedance and ion substitution techniques. Limitations of using the patch clamp for ion channel localization are presented.

Animals↗
PubMed1985

Steady state voltages in the frog lens.

Electro-chemical steady state in the lens depends on the transport properties of its various constituent cells. These transport properties, at a minimum, include the active transport of Na/K and the passive leak of Na, K and Cl through membrane channels. The work of Kinsey and Reddy (1), first localized active Na/K transport to the anterior surface cell membranes. In this paper, we estimate that the pump current density is 2 to 4 mu amp/cm2 of surface membrane, by measuring the change in intracellular voltage when the lens is exposed to 100 microM ouabain. Our impedance data suggest the passive leak of K is mostly across the membranes of surface cells, but whether these are anterior or posterior cells is not yet known. Membranes of the fiber cells throughout the volume of the lens appear to have channels that are selective for Na and Cl but few K channels. A simple model of electro-chemical steady state is derived to relate localized transport properties to the resting voltages in the lens. The above described localization of properties predicts radially circulating currents at steady state and spatial gradients in the intracellular and extracellular voltages. These predictions are compared to our measurements of steady state voltages and we find good agreement.

Animals↗
PubMed1984

Sutures of the crystalline lens: a review.

The sutures of the crystalline lens have previously been studied by light microscopy (LM). While the gross suture patterns (umbilical, line, y-shaped and star) of lenses are readily visualized by LM, fiber cell shape, curvature, length and the morphology of fiber cell ends cannot be adequately resolved by this technique. We have used scanning electron microscopy (SEM) to examine the sutures of crystalline lenses. SEM has revealed that in lenses with line, y-shaped or umbilical sutures, the anterior and posterior ends of fiber cells curve away in opposite directions from the polar axis of the lens before interlocking at suture branches. The degree of curvature decreases as a function of the number of suture branches. This relationship was not resolved by LM. SEM has revealed that the relationship of fiber cell taper to suture type was underestimated by previous LM studies. The reduction in fiber cell width from the equator to the sutures is 3:1 and 2:1 respectively, in lenses with line and y-shaped sutures. Furthermore, in lenses with star sutures, fiber cells are flared (1:1.7) rather than tapered, a fact not reported by previous LM studies. SEM also revealed that the offsetting of anterior and posterior suture branches does not result in equal fiber cell length in any growth ring as reported by LM studies. Rather, fiber cell length in any one growth ring varies as a sine wave function according to fiber cell location at the equator. Furthermore, the range of fiber cell length decreases as a function of the number of suture branches. Finally, SEM has revealed that the distal ends of fiber cells are expanded in both width and thickness prior to interlocking at suture branches and that these ends overlap rather than simply abut end-to-end to form a three dimensional suture plane extending down from the lens surfaces to the primary fiber cell mass.

Animals↗
PubMed1983

The electrical coupling of epithelium and fibers in the frog lens.

Electrical coupling has been measured between central epithelial cells and fiber cells in the frog lens following the removal of the lens capsule with collagenase. Current passed from a microelectrode inserted in a lens fiber cell was found to induce a potential difference in epithelial cells which was indistinguishable from that measured in nearby fiber cells. The measurements were made under circumstances where the current and voltage microelectrodes could be shown to be in different cells and an intraepithelial location of the voltage-measuring microelectrode could be verified. The electrical coupling appears to be direct from superficial fiber cells to epithelial cells, rather than indirect through equatorial epithelial cells. The epithelial cells and surface fiber cells each had resting potentials of -61.8 +/- 2.1 mV in this collagenase-treated preparation. Scanning electron microscopy (SEM) of mechanically decapsulated frog lenses showed specialized junction-like plaques on the expanded anterior ends of fiber cells which made contact with epithelial cells. Such specialized plaques were not found on the expanded posterior ends of these fibers where they contracted the posterior capsule. The studies provide direct evidence for cell-to-cell communication between frog lens epithelial cells and fibers.

Animals↗
PubMed1983

The use of procion dyes for light microscopy of the frog lens.

Protocols for light microscopy of frog lenses that result in good visibility of cell structure from the lens surface to its nucleus are presented. The lenses are fixed in 10% neutral formalin in 0.06 M phosphate buffer and are embedded in Epon 812. Following staining of the thick sections with selected Procion dyes, essentially every cell in the section plane of the lens can be visualized by simple light microscopy without fluorescence. The methods and dyes allow measurement of cell dimensions at all depths in the lens and allow investigation of cell packing geometry. The techniques should be generally useful for studying normal lens structure and the alteration of structure induced by cataractogenesis.

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