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Biomedical subjects

G L Fain

Publications and source records attributed to G L Fain.

At least 55 records · Page 3Linked to original sources
PubMed1988

Monoclonal antibodies to the H+-K+ ATPase of gastric mucosa selectively stain the non-pigmented cells of the rabbit ciliary body epithelium.

Monoclonal antibodies developed against the transport enzyme H+-K+ ATPase of gastric mucosa selectively bind to the nonpigmented cell layer in both the pars plana and pars plicata of the rabbit ciliary body epithelium. The most specific of these antibodies (HK 12.18) reacts with the nonpigmented cells but with no other cell type in ciliary body, retina or iris. Antibody reactivity is evinced by a distinct particulate staining which is greater on the surfaces of the nonpigmented cells than within the cytoplasm and seems most intense on the apical membrane between the pigmented and nonpigmented cell layers. This pattern of staining was present in 2-3-day-old pups and in young adults, in both pigmented and albino animals. Similar staining was not observed in control sections treated with preimmune serum, nonspecific mouse monoclonal antibody, or HK 12.18 preabsorbed to gastric microsomes. In addition to staining intact ciliary body, the antibody HK 12.18 can be shown to react with a population of cells lacking pigment granules in suspensions of dissociated ciliary body epithelium. This antibody may therefore be useful as a marker for the identification of nonpigmented cells in tissue culture.

Animals↗
PubMed1988

[3H]ouabain localization of Na-K ATPase in the epithelium of rabbit ciliary body pars plicata.

The secretion of the aqueous humor has been proposed to occur as the result of active Na+ transport by a ouabain-sensitive Na-K ATPase. We have examined the localization of this enzyme in the epithelium of rabbit ciliary body pars plicata using [3H]ouabain autoradiography. Single ciliary processes were isolated and incubated in Ringer containing [3H]ouabain. Processes were then rapidly frozen, freezedried, sectioned and exposed for autoradiography. In the light microscope, silver grains were found predominantly over the nonpigmented epithelial cells. In the electron microscope, grains could be localized for the most part to the interdigitations of the nonpigmented cell basolateral membrane. Label could also be observed at a much lower density above other membranes and above the pigmented and nonpigmented cell cytoplasm. No label was found in sections of control tissue which had been incubated in [3H]ouabain with an excess of cold ouabain. To show that the [3H]ouabain had free access to all of the membrane surfaces within the epithelium, in parallel experiments we incubated isolated processes in horseradish peroxidase. Our experiments suggest that most of the active Na+ transport in ciliary body epithelium occurs across the basolateral membrane of nonpigmented cells into the posterior chamber. Furthermore, the placement of the Na-K ATPase within the narrow membrane infoldings of the interdigitations is consistent with a role for this enzyme in water transport and the production of the aqueous.

Animals↗
PubMed1988

Isolation of non-pigmented epithelial cells from rabbit ciliary body.

We describe a new technique for the separation and isolation of nonpigmented ciliary body epithelial cells from rabbit. Excised ciliary body is incubated in a medium buffered with EGTA to a free-Ca2+ concentration of 10(-8) M, and the nonpigmented cell layer is separated from the pigmented layer by microdissection under direct visualization. This technique yields intact cells which are greater than 99% nonpigmented. It can be used to produce preparations of nonpigmented cell plasma membrane and of viable whole cells, which may be useful for biochemistry, pharmacology, cell transport studies and tissue culture.

Animals↗
PubMed1987

Calcium in dark-adapted toad rods: evidence for pooling and cyclic-guanosine-3'-5'-monophosphate-dependent release.

1. We have used laser micromass analysis (l.a.m.m.a.) to investigate Ca uptake and release in intact 'red' rod photoreceptors in the dark-adapted retina of the toad, Bufo marinus. 2. With l.a.m.m.a. it is possible to measure separately the concentrations of each of the Ca isotopes. Rods normally containing almost exclusively 40Ca can be incubated in Ringer solution containing the stable isotopes 42Ca or 44Ca. In this way, the movements of Ca into and out of the rod can be separately determined. 3. When rods are incubated in darkness in high 44Ca (up to 20 mM), large amounts of 44Ca accumulate in the outer segment at a rate which increases with increasing external 44Ca concentration. However, this 44Ca appears not to exchange with the 40Ca originally present within the rod. This result suggests that the 40Ca may be sequestered within a pool which normally exchanges slowly with external Ca. 4. We explored Ca exchange in high-Ca solutions in more detail with double-isotope labelling. In these experiments, rods were first pre-loaded with Ca of one isotope (42Ca) and then incubated in Ringer solution containing another (44Ca). We could then measure separately the rate of exchange of the pre-loaded 42Ca with the 44Ca in the Ringer solution and with the 40Ca originally present within the rod in the sequestered pool. 5. These experiments show that the pre-loaded-Ca exchanges rapidly with Ca in the Ringer solution, at least in part by Ca-Ca exchange, but much more slowly with the Ca originally present within the rod. Thus Ca in the outer segments can exist in (at least) two pools: one which exchanges rapidly across the plasma membrane and is probably Ca free or loosely bound within the cytosol, and another which exchanges slowly and is probably Ca within the disks. 6. Although Ca sequestered within the outer segment normally exchanges quite slowly, it can be rapidly released if the extracellular free Ca is buffered to low levels with EGTA. The rate-limiting step for Ca release under these conditions appears not to be Na-Ca exchange, since the rate of Ca efflux is unchanged if the Na in the Ringer solution is substituted with choline. 7. Ca can also be released from the sequestered pool if rods are incubated in Ringer solution containing 100 or 500 microM-IBMX (3-isobutyl-1-methylxanthine).(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1987

Volume regulation of non-pigmented cells from ciliary epithelium.

We describe a new method for investigating ion and water transport in ciliary epithelium. A single ciliary process from the rabbit is isolated, placed in a chamber, and rapidly perfused with physiological Ringer. If this process is then viewed at its edge with a light microscope using Hoffman modulation contrast optics, it is possible to record the image of a single layer of non-pigmented epithelial cells. When these cells are exposed to hypotonic Ringer, they swell and then reduce their volume by extruding salt and water. We have used the rate of swelling to calculate the hydraulic conductivity of the non-pigmented cells. These measurements show that the aqueous humor could be secreted by the generation of a modest osmotic gradient across the non-pigmented cell basal membrane. We have also used this preparation to investigate the mechanism of the decrease in cell volume after swelling in hypotonic medium. This volume regulatory decrease can occur at a rate as large as the rate of normal fluid transport during the secretion of the aqueous. It appears to be caused in part by active Na extrusion, since it is partially inhibited by exposure to ouabain. In addition, there appears also to be a contribution to the regulatory response from a swelling-induced K efflux, since the rate of volume regulation can be altered by perfusion with Ba2+ and by changing the extracellular K concentration.

Animals↗
PubMed1985

Calcium content and calcium exchange in dark-adapted toad rods.

We have used laser-activated micro mass analysis (l.a.m.m.a.) and energy-dispersive X-ray analysis (e.d.x.) to measure Ca content and Ca movements in 'red' rod photoreceptors in the dark-adapted retina of the toad, Bufo marinus. Measurements with both l.a.m.m.a. and e.d.x. show that intact rod outer segments contain 4-5 mmol total Ca/l wet tissue volume, or 1-2 Ca per rhodopsin. We could detect no significant variation in the total Ca as a function of distance across or up and down the outer segment. In the inner segment, Ca could be detected only within the mitochondria-rich ellipsoid body, where the total Ca concentration was of the order of 100-400 mumol/l wet tissue volume. To measure the exchange of Ca in outer segments from intact photoreceptors, we exposed the dark-adapted retina to Ringer containing the stable isotope 44Ca. Since l.a.m.m.a. can measure separately the concentrations of each of the isotopes of the elements, and since native rods contain almost exclusively 40Ca, the increase in 44Ca and decrease in 40Ca could be used as a measure of Ca influx and efflux. Ca exchange in intact rod outer segments in darkness is very slow. The rate of accumulation of 44Ca was only 10(5) Ca/rod.s, or about 10% of the total outer segment Ca/h. This slow rate of exchange is apparently not the result of restricted movement of Ca across the plasma membrane. Ca exchange was also measured in outer segments which were either partially or entirely detached from the rest of the photoreceptor. In broken-off outer segments, Ca exchange is faster than in the intact organelles, and in 1 h, half of the 44Ca exchanges for 40Ca. When the retina was incubated in Ringer for which all of the Na was substituted with Li or choline, there was an increase in the rate of 44Ca accumulation in intact outer segments, probably due to an inhibition of Na-Ca counter transport across the plasma membrane. Our measurements indicate that the great majority of the Ca in the rod appears to be inaccessible to exchange under physiological conditions, probably because it is sequestered within the disks which in intact rods appear to be nearly impermeable to Ca in darkness.

Animals↗
PubMed1983

Mechanisms of synaptic transmission in the retina.

This paper reviews the mechanisms of transmitter release, the kinetics of synaptic transfer, the mechanisms for the production of conductance changes by transmitters, and the nature of the conductance changes at synapses in vertebrate retina. A method for the culturing of adult retinal cells is described, together with preliminary experiments on the identification of cells in culture.

Animals↗
PubMed1982

Voltage-dependent conductances in Limulus ventral photoreceptors.

The voltage-dependent conductances of Limulus ventral photoreceptors have been investigated using a voltage-clamp technique. Depolarization in the dark induces inward and outward currents. The inward current is reduced by removing Na+ or Ca2+ and is abolished by removing both ions. These results suggest that both Na+ and Ca2+ carry voltage-dependent inward current. Inward current is insensitive to tetrodotoxin but is blocked by external Ni2+. The outward current has a large transient component that is followed by a smaller maintained component. Intracellular tetraethylammonium preferentially reduces the maintained component, and extracellular 4-amino pyridine preferentially reduces the transient component. Neither component is strongly affected by removal of extracellular Ca2+ or by intracellular injection of EGTA. It is concluded that the photoreceptors contain at least three separate voltage-dependent conductances: 1) a conductance giving rise to inward currents; 2) a delayed rectifier giving rise to maintained outward K+ current; and 3) a rapidly inactivating K+ conductance similar to the A current of molluscan neurons.

4-Aminopyridine↗
PubMed1982

Light-dependent ion influx into toad photoreceptors.

To measure the influx of Na+ and other ions through the light-dependent permeability of photoreceptors, we superfused the isolated retina of the toad, Bufo marinus, with a low-Ca2+ (10(-8) M), low-Cl- Ringer's solution containing 0.5 mM ouabain. Under these conditions, the membrane potential of the rod is near zero and there is no light-induced potential change either in the rod or in more proximal neurons. The photoreceptors, however, continue to show a light-dependent increase in membrane resistance, which indicates that the light-sensitive channels still close with illumination. Dark-adapted retinas show a larger 22Na+ accumulation than do light-adapted retinas. The extra accumulation of 22Na+ into dark-adapted retinas can be removed if the retinas are washed in darkness with low-Ca2+ Ringer's solutions, or if the ionophore gramicidin D is present in the perfusate. The additional accumulation in dark retinas corresponds to a flux of at least 10(9) Na+ per receptor per second, which is close to the value of the photoreceptor dark current. The light-dependent uptake of 22Na+ can be prevented by exposing the retinas to Ca2+ during the incubation period, but is restored if the phosphodiesterase inhibitor IBMX is added to the perfusate. A significant light-dependent ion accumulation can be observed for the cations K+, Rb+, Cs+, and Tl+, in addition to Na+, but not for methylamine, choline, or tetraethylammonium.

1-Methyl-3-isobutylxanthine↗
PubMed1982

Ca2+-dependent changes in cyclic GMP levels are not correlated with opening and closing of the light-dependent permeability of toad photoreceptors.

We have measured the levels of 3',5'-guanosine monophosphate (cyclic GMP) in isolated retinas from toad to investigate their correlation to the opening and closing of the light-dependent permeability of photoreceptors. When Ca2+-induced changes in cyclic GMP concentration are compared with the Ca2+-induced changes in the permeability of photoreceptor light-dependent channel, four quantitative dissimilarities are noted. First, when extracellular Ca2+ ([Ca2+]o) is reduced from normal physiological levels to between 10(-6) and 10(-7) M, the light-dependent permeability is increased, but cyclic GMP levels are not significantly changed. Second, when [Ca2+]o is increased from 1.8 to 20 mM, the light-dependent permeability is suppressed, but cyclic GMP levels are decreased by only 10-15%, about one-quarter the decrease that can be obtained with bright illumination. Third, when [Ca2+]o is increased from 10(-8) M to 20 mM, the light-dependent permeability is closed rapidly, but the cyclic GMP decrease is slow. Fourth, when [Ca2+]o is lowered to 10(-8) M, the sensitivity of the light-dependent permeability to steady illumination is decreased by three to four orders of magnitude, but the sensitivity of the light-dependent decrease in cyclic GMP is not significantly affected. These observations indicate that there is no simple correlation between cyclic GMP levels and the permeability of the light-dependent channels and that Ca2+ can affect the conductance in the absence of changes in cyclic GMP content.

Animals↗
PubMed1982

The effects of low calcium and background light on the sensitivity of toad rods.

1. We have examined the effects of decreases in extracellular Ca(2+) concentration on the intracellularly recorded light responses of rods from the toad, Bufo marinus. In agreement with previous results (Brown & Pinto, 1974; Lipton, Ostroy & Dowling, 1977), Ca(2+) concentrations below 10(-6) M produced a depolarization of rod resting membrane potential of approximately 30-40 mV and a corresponding increase in the maximum amplitude of the rod's light responses, so that saturating flashes in normal and low Ca(2+) Ringer produced hyperpolarizations to approximately the same membrane potential.2. The rod's sensitivity was reduced in low Ca(2+) Ringer by an amount dependent upon the extracellular Ca(2+) concentration. At 10(-6) M-Ca(2+), sensitivity was approximately 0.6 log units below normal. Thereafter, it dropped nearly linearly with [Ca(2+)](o) to a value approximately 4.0 log units below normal at 10(-9) M-Ca(2+). Most of the decline occurred within 1-2 min after the solution change as the membrane potential depolarized, but sensitivity continued to fall slowly with time at the lowest Ca(2+) concentrations. Exposure to low Ca(2+) solutions altered the kinetics of the receptor response to brief flashes, delaying response onset and time-to-peak but affecting the time course of decay very little.3. The sensitivity of the rod to maintained steps of light was also reduced in low Ca(2+). Furthermore, the changes in sensitivity produced by background illumination were very much smaller in low Ca(2+) than in normal Ringer. In some cases backgrounds actually increased sensitivity.4. In 10(-8) M-Ca(2+), backgrounds which themselves produced no response in the rod and no changes in rod sensitivity produced large decreases in response latency for responses of all amplitudes, and pronounced changes in time-to-peak and time-to-decay for moderate and large amplitude responses.5. Since the effects of background light and low Ca(2+) on the wave form of the rod are distinct and in some cases antagonistic, and since the changes in receptor sensitivity produced by backgrounds and low Ca(2+) are not additive, the decreases in sensitivity produced by exposure to low Ca(2+) appear to be caused by a mechanism distinct from normal light adaptation. We suggest that they are caused by an increase in the buffering capacity of the receptor cytosol for Ca(2+) and that Ca(2+) is the excitatory messenger or ;internal transmitter', as originally suggested by Yoshikami & Hagins (1971).

Adaptation, Ocular↗
PubMed1982

The effects of sodium replacement on the responses of toad rods.

1. We have investigated the effects of Na(+) substitution on the membrane potential and light responses of rods in the superfused retina of the toad, Bufo marinus.2. When all of the Na(+) in the Ringer was replaced with Li(+), the effects on the rods depended upon the external free Ca(2+) concentration ([Ca(2+)](o)). At [Ca(2+)](o) >/= 10(-6) M, the membrane potential (E(m)) hyperpolarized and light responses were greatly diminished or abolished. At [Ca(2+)](o) </= 10(-7) M, Li(+) replacement had little effect on E(m) or response amplitude.3. We interpret these results as revealing a Na(+)-Ca(+) counter-transport in rods. At high [Ca(2+)](o), replacing Na(+) with Li(+) would have produced an increase in the rod cytosol free Ca(2+) concentration ([Ca(2+)](i)) and the blockage of the light-dependent conductance, leading directly to the suppression of light responses. At [Ca(2+)](o) </= 10(-7) M, this presumably would not have occurred.4. Since at these low Ca(2+) concentrations we observed light responses of nearly normal amplitude in Li(+), our results suggest that the light-dependent conductance is permeable to Li(+).5. Substitution of Na(+) with K(+) in low Ca(2+) produced a complete suppression of the responses. However, it was still possible to measure large light-induced changes in rod input resistance.6. Substitution of Na(+) with tetramethylammonium, tetraethylammonium, Tris, or choline in low Ca(2+) produced a large hyperpolarization of the membrane potential and a diminution of response amplitude. However, we were unable to observe a complete suppression of the responses for these cations.7. Substitution of Na(+) with tetrapropylammonium or with an uncharged substance (glucose or urea) in low Ca(2+) produced a large hyperpolarization of membrane potential and a considerable decrease in the light responses. In about half our attempts, the responses were observed to decline reversibly to less than 20% of their peak amplitude in Na(+).8. Results with tetrapropylammonium were indistinguishable from those of glucose or urea, indicating that the light-dependent conductance probably is not permeable to TPA. The resistance changes measured with K(+) substitution and the responses observed in the presence of the organic ions TMA, TEA, Tris and choline suggest that these species may be permeable, but we are unable to discount alternative explanations.

Animals↗
PubMed1981

D-aspartate potentiates the effects of L-glutamate on horizontal cells in goldfish retina.

The amino acids L-glutamate and L-aspartate depolarize H1 horizontal cells in the perfused goldfish retina but only at millimolar concentrations. The effects of L-glutamate (but not of L-aspartate) are potentiated approximately 15-fold by exposure to D-aspartate. D-Aspartate blocks acidic amino acid uptake in goldfish retina, so that the potentiation of L-glutamate may be produced by an increase in its effective concentration at the horizontal cell membrane. Because D-aspartate also augments the light responses of horizontal cells, our results are consistent with the possibility that L-glutamate is a neurotransmitter of cone photoreceptors in goldfish.

Animals↗
PubMed1980

Calcium spikes in toad rods.

1. When the retina of the toad, Bufo marinus, was superfused with 6-12 mM-tetraethylammonium chloride (TEA), intracellular recordings from rods showed large, depolarizing regenerative potentials. For brief exposures to TEA, these potentials occurred during the recovery phase of the light responses; whereas, during longer exposures, they were spontaneous in darkness but suppressed during illumination. Similar regenerative potentials were observed during perfusion with 3-10 mM-4-aminopyridine and 1-2 mM-BaCl2. 2. The amplitude of the regenerative potentials depended upon the extracellular Ca concentration ([Ca2+]o). Lowering [Ca2+]o decreased their amplitude and in zero [Ca2+]o they were reversibly abolished. Increasing [Ca2+]o by 1.5-2 times produced a small hyperpolarization of membrane potential and a large augmentation in regenerative response amplitude. However, larger increases in [Ca2+]o produced large membrane hyperpolarizations and reversibly suppressed the regenerative responses. 3. High concentrations of Sr2+ in TEA also enhanced regenerative activity but did not affect the rod resting membrane potential. The amplitude of regenerative potentials increased continuously with increasing [Sr2+]o, and in 28 mM-Sr2+ the rods generated 60-70 mV action potentials, even in the absence of extracellular Na+. 4. The regenerative potentials were blocked by 25 microM-Cd2+, 50-100 microM-Co2+, 5mM-Mg2+, and 100 microM-D-600. They were unaffected by 2 microM-TTX or 2-5 mM-Na aspartate. 5. In Ringer containing 12 mM-TEA, large anode break responses could be recorded from rods at the termination of inward current pulses. These anode break responses were also suppressed by Co2+ and unaffected by TTX or Na aspartate. 6. We conclude that the membrane of toad rods contains a conductance normally selective for Ca2+, which is activated by depolarization. In normal Ringer, the inward current through this conductance produces little effect, since it is balanced by a large outward current, probably carried by K+. TEA and other agents appear to block this outward current, permitting the Ca2+ current to become regenerative.

Action Potentials↗
PubMed1980

The effects of tetraethylammonium and cobalt ions on responses to extrinsic current in toad rods.

1. Double-barrel micropipettes were used to pass pulses of current in darkness into single rods in the isolated, perfused retina of the toad, Bufo marinus. 2. In normal Ringer solution, current pulses evoked non-linear changes in membrane potential which varied as a function of current amplitude and of time. Responses to currents of both polarities showed slow relaxations toward the base line during the pulse, and the steady-state I-V curve exhibited a prominent outward rectification. 3. In Ringer containing 12 mM-TEA, the slow relaxation of voltage during outward current pulses was diminished, and the outward rectification was markedly reduced. In contrast Co2+, at a concentration in excess of that required to block Ca2+ spikes in rods, increased the receptor input resistance but did not reduce either the amplitude of the slow relaxation or the extent of outward rectification. 4. These experiments indicated that the outward rectification of rods is predominantly due to a conductance which is gated by voltage rather than by entry of Ca2+. 5. Long-lasting after-potentials followed the termination of outward current pulses. In normal Ringer the after-potentials were hyperpolarizing and were accompanied by an increase in input conductance. In TEA, the afterpotentials were depolarizing and were also accompanied by an increase in input conductance. The after-depolarizations in TEA were enhanced by Sr2+ and blocked by Co2+. These experiments suggest that the hyperpolarizing and depolarizing afterpotentials are produced by different mechanisms, the hyperpolarizing by an increase in K+ conductance, and the depolarizing by an increase in Ca2+ conductance.

Animals↗
PubMed1979

Light adaptation in toad rods: requirement for an internal messenger which is not calcium.

1. The mechanism of light adaptation was investigated by recording intracellularly from single rods in the isolated, superfused retina of the toad, Bufo marinus. Steady background lights produce decreases in rod sensitivity and changes in response wave form similar to those previously observed in the toad eyecup. 2. The sensitivity of a dark-adapted rod is halved by a background light which bleaches about 4 rhodopsins per rod per second. Since a toad rod contains over 2000 disks, a rhodopsin bleached in one disk must alter the effectiveness of rhodopsins bleached in others. This could occur if the state of adaptation in the rod were regulated by the concentration of some diffusable substance. 3. This diffusable substance cannot be Ca2+. Increases in intracellular Ca2+, produced experimentally either by increasing extracellular Ca2+ or by facilitating Ca2+ permeability into the rod with the ionophore X537A, cause a hyperpolarization of membrane potential and a decrease in response amplitude; but they do not produce changes in sensitivity and response wave form like those produced by background light. 4. Either Ca2+ is not the internal transmitter released from the disks during excitation, or the disks release or otherwise alter the concentration of a second diffusable substance, in addition to Ca2+, which regulates the state of adaptation.

Adaptation, Ocular↗