Biomedical subjects

G L Fain

Publications and source records attributed to G L Fain.

At least 19 recordsLinked to original sources

PubMed1999

Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Ambystoma↗

PubMed1998

Bleached pigment produces a maintained decrease in outer segment Ca2+ in salamander rods.

A spot confocal microscope based on an argon ion laser was used to make measurements of cytoplasmic calcium concentration (Ca2+i) from the outer segment of an isolated rod loaded with the fluorescent calcium indicator fluo-3 during simultaneous suction pipette recording of the photoresponse. The decline in fluo-3 fluorescence from a rod exposed to saturating illumination was best fitted by two exponentials of approximately equal amplitude with time constants of 260 and 2,200 ms. Calibration of fluo-3 fluorescence in situ yielded Ca2+i estimates of 670 +/- 250 nM in a dark-adapted rod and 30 +/- 10 nM during response saturation after exposure to bright light (mean +/- SD). The resting level of Ca2+i was significantly reduced after bleaching by the laser spot, peak fluo-3 fluorescence falling to 56 +/- 5% (SEM, n = 9) of its value in the dark-adapted rod. Regeneration of the photopigment with exogenous 11-cis-retinal restored peak fluo-3 fluorescence to a value not significantly different from that originally measured in darkness, indicating restoration of the dark-adapted level of Ca2+i. These results are consistent with the notion that sustained activation of the transduction cascade by bleached pigment produces a sustained decrease in rod outer segment Ca2+i, which may be responsible for the bleach-induced adaptation of the kinetics and sensitivity of the photoresponse.

Adaptation, Ocular↗

PubMed1998

Synergistic receptor-activated calcium increases in single nonpigmented epithelial cells.

PURPOSE: To determine whether single nonpigmented ciliary body cells contain the signaling mechanism to produce synergistic drug-activated increases in Ca2+, or whether these responses are produced cooperatively by interaction among groups of cells. METHODS: Suspensions of single nonpigmented cells were plated onto soft collagen gels. Fura-2 fluorescence ratio imaging was used to examine receptor-evoked changes in intracellular Ca2+ concentration. RESULTS: Nonpigmented cells plated on soft collagen gels retained a rounded shape with membrane evaginations visible on their surface. Application of acetylcholine (10 microM) or epinephrine (1 microM) each produced small increases in intracellular Ca2+, but in combination they produced a Ca2+ increase of more than 10-fold. This synergistic Ca2+increase was a result of activation of muscarinic and alpha2-adrenergic receptors because a specific alpha2-adrenergic agonist could substitute for epinephrine in producing the response. The response could be blocked by a specific alpha2-antagonist and a muscarinic antagonist. An alpha1-agonist could not substitute for epinephrine in producing a synergistic increase nor could the synergism be blocked by alpha1- or beta-antagonists. The Ca2+ increase was largely produced by release from internal stores, because the peak amplitude of the response was nearly the same in the external solution containing a low Ca2+ concentration; however, the influx of Ca2+ into the cell was responsible for maintenance of a steady component of the Ca2+ increase during maintained drug stimulation and for refilling the internal stores. CONCLUSIONS: Single nonpigmented cells can produce synergistic increases in Ca2+ on multiple receptor activation, indicating that the mechanism of synergism does not require the interaction of multiple cells. The Ca2+ increase is a result of release from internal stores and Ca2+ entry through an as yet undefined conductance or transport system in the plasma membrane.

Acetylcholine↗

PubMed1997

Synergistic rise in Ca2+ produced by somatostatin and acetylcholine in ciliary body epithelial cells.

The purpose of these experiments was to demonstrate the presence of somatostatin receptors on the nonpigmented epithelial cells of the rabbit ciliary body and their link with intracellular Ca2+ homeostasis. Freshly excised rabbit ciliary processes and nonpigmented cell layer, explants were loaded with the fluorescent dye fura-2, and free-Ca2+ concentration ([Ca2+]i) in the nonpigmented cells was measured with fluorescence ratio imaging. The cells were continuously perfused, and drugs were added to the perfusate. Somatostatin-14 (SS14, 0.1-1.0 microM) or acetylcholine (ACh, 10 microM) applied alone produced small increases in [Ca2+]i. However, SS14 (0.1 microM) in combination with ACh (10 microM) induced a massive increase in [Ca2+]i (25.7 +/- 3.3 times the baseline level, n = 28). The dose-response curve for SS14 (in the presence of 10 microM ACh) was sigmoidal with an EC50 of 3.9 nM and Hill coefficient of 2.5, indicating the requirement for multiple SS receptor activation. Somatostatin-28 could mimic the effect of SS14, although a much higher concentration was required. Shifting the SS14 dose-response curve to the right by about two-orders of magnitude resulted in a fit to the SS28 data. The response to ACh + SS14 could not be blocked by the alpha 2-adrenergic blocker yohimbine (Yoh, 10 microM) or the A1-specific adenosinergic antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 1 microM). Incubation of the tissue with pertussis toxin (PTx, 1 microgram ml-1) did not alter the response to ACh alone but eliminated the synergistic effect of somatostatin. We conclude that nonpigmented epithelial cells of the rabbit ciliary body possess a novel somatostatin receptor whose activation can synergistically potentiate the rise in [Ca2+]i produced by ACh. This potentiation appears to occur via a pertussis-toxin-sensitive pathway, perhaps through Gi.

Acetylcholine↗

PubMed1996

Starburst amacrine cells change from spiking to nonspiking neurons during retinal development.

The membrane excitability of cholinergic (starburst) amacrine cells was studied in the rabbit retina during postnatal development. Whole-cell patch-clamp recordings were made from 110 displaced starburst cells in a thin retina] slice preparation of rabbits between postnatal days P1 and P56 old. We report that displaced starburst cells undergo a dramatic transition from spiking to nonspiking, caused by a loss of voltage-gated Na currents. This change in membrane excitability occurred just after eye opening (P10), such that all of the starburst cells tested before eye opening had conspicuous tetrodotoxin-sensitive Na currents and action potentials, but none tested after the first 3 postnatal weeks had detectable Na currents or spikes. Our results suggest that starburst cells use action potentials transiently during development and probably play a functional role in visual development. These cells then cease to spike as the retina matures, presumably consistent with their role in visual processing in the mature retina.

Action Potentials↗

PubMed1996

Role of cytoplasmic calcium concentration in the bleaching adaptation of salamander cone photoreceptors.

1. In order to study the possible involvement of Ca2+ in the bleaching adaptation of cones isolated from the retina of the salamander Ambystoma tigrinum, changes in cytoplasmic calcium concentration ([Ca2+]i were opposed by exposing the outer segment to a low-Ca(2+)-O Na+ solution designed to minimize Ca2+ fluxes across the outer segment membrane. 2. When a cone was exposed in normal Ringer solution to bright light bleaching a significant fraction of the photopigment, the circulating current was initially suppressed completely and then recovered to a maintained value less than the value in darkness before the bleach. When the outer segment of the cone was stepped to low-Ca(2+)-O Na+ solution before the bleach was delivered, the circulating current recovered more slowly or (for large bleaches) remained completely suppressed for the duration of the solution exposure. 3. If, during the period for which the current was suppressed in low-Ca(2+)-O Na+ solution, the cone outer segment was exposed to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), the circulating current was restored. The dim flash response recorded under these conditions exhibited kinetics and integration times similar to those recorded in low-Ca(2+)-O Na+ solution in darkness before the bleach. If, instead, the outer segment was returned to Ringer solution after the bleach, thereby allowing [Ca2+]i to fall from its dark-adapted level to the appropriate bleach-adapted level, the kinetics of the response in low-Ca(2+)-O Na+ solution were greatly accelerated, and the integration time considerably reduced. This was true regardless of whether or not the low-Ca(2+)-O Na+ solution included IBMX. 4. The role of Ca2+ in bleaching adaptation appeared to resemble its role in background adaptation, since in both cases exposure to low-Ca(2+)-O Na+ solution suppressed the acceleration of response kinetics. Responses recorded from cones in low-Ca(2+)-O Na+ solution were nearly identical in waveform and sensitivity during background light or after bleaches, provided that IBMX was used to restore sufficient photocurrent so that responses to flashes could be recorded, and sensitivity was corrected for loss in quantum catch. 5. These results indicate that the fall in [Ca2+]i in cones after a bleach is necessary both for the acceleration of the flash response and the adaptational decrease in sensitivity, as is the case for adaptation by background light.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Dark adaptation in vertebrate photoreceptors.

Exposure of the eye to bright light bleaches a significant fraction of the photopigment in rods and cones and produces a prolonged decrease in the sensitivity of vision, which recovers slowly as the photopigment is regenerated. This sensitivity decrease is larger than would be expected merely from the decrease in the concentration of the pigment. Recent experiments have shown that the decrease in sensitivity is produced largely by an excitation of the phototransduction cascade by bleached pigment; even in darkness, it produces an equivalent background similar to that produced by real steady background illumination. Thus, excitation produced by a form of rhodopsin thought previously to be inactive has a profound effect on the physiology of the photoreceptor. This raises the possibility that forms of other G protein-coupled receptors thought to be inactive might also play an important role in signal transduction and disease.

Adaptation, Physiological↗

PubMed1996

Equivalence of background and bleaching desensitization in isolated rod photoreceptors of the larval tiger salamander.

Psychophysical experiments have shown an equivalence between sensitivity reduction by background light and by bleaches for the human scotopic system. We have compared the effects of backgrounds and bleaches on the light-sensitive membrane-current responses of isolated rod photoreceptors from the salamander Ambystoma tigrinum. The quantum catch loss was factored out from the desensitization due to bleaching to give the fraction of "extra" desensitization due to adaptation. For backgrounds, desensitization is well described by the Weber/Fechner equation. The extra desensitization after bleaches can also be described by the Weber/Fechner equation, if an "equivalent" background produced by bleaching is made linearly proportional to the fraction of pigment bleached. A background which produces an extra desensitization of a factor of two is equivalent to a fractional bleach of approximately 6%. Equivalent background and bleaching desensitizations were associated with similar reductions in circulating current. There is a linear relation between log flash sensitivity and decrease in circulating current. Equivalent background and bleaching desensitizations were associated with similar increases in cGMP phosphodiesterase and guanylate cyclase activity. These were inferred from membrane current changes after steps into lithium or IBMX solutions. There were also similar reductions in the integration times of dim flash responses for equivalent desensitizations produced by backgrounds and bleaches. These results suggest that the equivalence between background and bleaching found psychophysically may arise at the very earliest stages of visual processing and that these two processes of desensitization have similar underlying mechanisms.

Adaptation, Physiological↗

PubMed1996

Persistent activation of transducin by bleached rhodopsin in salamander rods.

The hydrolysis-resistant GTP analogue GTP-gamma-S was introduced into rods isolated from the retina of the salamander Ambystoma tigrinum to study the origin of the persistent excitation induced by intense bleaching illumination. Dialysis of a dark-adapted rod with a whole-cell patch pipette containing 2 mM GTP-gamma-S resulted in a gradual decrease in circulating current. If the rod was first bleached and its sensitivity allowed to stabilize for at least 30 min, then dialysis with GTP-gamma-S produced a much faster current decay. The circulating current could be restored by superfusion with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting that the decay in current originated from persistent excitation of the phosphodiesterase by transducin bound to GTP-gamma-S. We conclude that the persistent excitation which follows bleaching is likely to involve the GTP-binding protein transducin, which mediates the normal photoresponse. This observation suggests that a form of rhodopsin which persists long after bleaching can activate transducin much as does photoisomerized rhodopsin, although with considerably lower gain.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Bleached pigment activates transduction in salamander cones.

We have used suction electrode recording together with rapid steps into 0.5 mM IBMX solution to investigate changes in guanylyl cyclase velocity produced by pigment bleaching in isolated cones of the salamander Ambystoma tigrinum. Both backgrounds and bleaches accelerate the time course of current increase during steps into IBMX. We interpret this as evidence that the velocity of the guanylyl cyclase is increased in background light or after bleaching. Our results indicate that cyclase velocity increases nearly linearly with increasing percent pigment bleached but nonlinearly (and may saturate) with increasing back-ground intensity. In cones (as previously demonstrated for rods), light-activated pigment and bleached pigment appear to have somewhat different effects on the transduction cascade. The effect of bleaching on cyclase rate is maintained for at least 15-20 min after the light is removed, much longer than is required after a bleach for circulating current and sensitivity to stabilize in an isolated cone. The effect on the cyclase rate can be completely reversed by treatment with liposomes containing 11-cis retinal. The effects of bleaching can also be partially reversed by beta-ionone, an analogue of the chromophore 11-cis-retinal which does not form a covalent attachment to opsin. Perfusion of a bleached cone with beta-ionone produces a rapid increase in circulating current and sensitivity, which rapidly reverses when the beta-ionone is removed. Perfusion with beta-ionone also causes a partial reversal of the bleach-induced acceleration of cyclase velocity. We conclude that bleaching produces an "equivalent background" excitation of the transduction cascade in cones, perhaps by a mechanism similar to that in rods.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Neurotransmitter receptors of starburst amacrine cells in rabbit retinal slices.

The receptor pharmacology of cholinergic ("starburst") amacrine cells was studied in a newly developed rabbit retinal slice preparation with whole-cell patch clamp. Displaced starburst cells were labeled with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI), and their dendritic morphology was identified in the slice with Lucifer yellow. Under conditions in which synaptic transmission was blocked by Cd2+, starburst cells responded vigorously to the bath-applied neurotransmitters GABA, glycine, and glutamate. The response to GABA consisted of an inward current and an increase in noise, which could be mimicked by the GABAA agonists muscimol and trans-4-aminocrotonic acid (TACA), but not by the GABAB agonist baclofen or the GABAC agonist cis-4-aminocrotonic acid (CACA). The GABA-evoked currents were reversibly inhibited by bicuculline and picrotoxin and had a reversal potential close to the chloride equilibrium potential. Noise analysis of GABA-activated whole-cell currents yielded elementary conductance estimates of 12.5 pS. Glycine (30-200 microM) also activated a Cl- conductance in starburst cells, which could be completely blocked by strychnine. The non-NMDA agonists kainate (KA, 30-100 microM) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 60 microM) evoked robust responses, which were reversibly blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and which reversed near the equilibrium potential for cations. NMDA coapplied with glycine in salines free of Cd2+ and Mg2+ elicited small but detectable responses. The I/V relation of the NMDA-evoked response showed a characteristic "J"-shaped region in a saline containing 1 mM Mg2+ and 0 Cd2+, indicating that NMDA receptors were present directly on starburst cells. This was consistent with our finding that whole-cell currents evoked by KA and NMDA had different noise characteristics. These results place new constraints on models of starburst cell function and suggest that GABA-mediated inhibition of the starburst cell itself may play an important role in directional selectivity in the retina.

Animals↗

PubMed1994

Bleached pigment activates transduction in isolated rods of the salamander retina.

1. We have used suction electrode recording together with rapid steps into Li+ solution and 0.5 mM IBMX solution to estimate the rates of the guanylyl phosphodiesterase (PDE) and guanylyl cyclase in isolated rods of the salamander, Ambystoma tigrinum. 2. We show that both the PDE and cyclase velocities are accelerated by steady background light. The steady velocities of both enzymes appear to be saturating functions of background intensity. 3. Bleaching also accelerates both the PDE and cyclase. This effect is maintained long after the bleaching stimulus is removed (up to 2 h) and is reversed only if the photopigment is regenerated with exogenous chromophore. 4. The estimated steady-state PDE and cyclase velocities appear to be linear functions of the amount of pigment bleached, as if each bleached pigment molecule activated the transduction cascade with the same probability and gain. 5. The effectiveness of bleached pigment in activating transduction is only 10(-6) to 10(-7) times that of activated rhodopsin (Rh*), but this is sufficient after large bleaches to produce an 'equivalent background' excitation of the rod, which is probably responsible, at least in part, for bleaching desensitization.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Amino acid receptors of midget and parasol ganglion cells in primate retina.

Primate retinas contain two major ganglion cell types. Midget (or P type) cells have relatively sustained responses to light; the amplitude and polarity of these responses vary with stimulus wavelength. Parasol (or M type) cells are more sensitive to stimulus contrast and respond more transiently but are not selective for color. Both types can be further subdivided into a and b subtypes, according to the level of their dendritic stratification in the inner plexiform layer. To determine whether differences in receptors for amino acid transmitters are the basis for any differences in ganglion cell light responses, we made whole-cell, patch-clamp recordings from identified ganglion cells in slice preparations of macaque and baboon retinas. We found that midget and parasol cells of both a and b types had similar responses to excitatory amino acids, including kainate, alpha-amino-3-hydroxy-5-methylisoxalzole-4-propionic acid, and N-methyl-D-aspartate, with reversal potentials near the equilibrium potential for cations. Kainate responses were blocked by 6,7-dinitroquinoxaline, and N-methyl-D-aspartate responses were blocked by D-(-)-2-amino-7-phosphonoheptanoic acid. The four types of ganglion cells also had similar responses to bath-applied inhibitory amino acids. All cells had both gamma-aminobutyric acid and glycine receptors with reversal potentials near the equilibrium potential for Cl-, and the relative amplitudes of the responses to excitatory and inhibitory amino acids were similar among the various cell types. These results suggest that the differences in response properties of the different classes of ganglion cells in primate retina may be determined, to a significant degree, by the properties of the amacrine and bipolar cells that provide their input rather than by the nature of their postsynaptic receptors.

Animals↗

PubMed1994

Dihydropyridine-sensitive Ca2+ spikes and Ca2+ currents in rabbit ciliary body epithelial cells.

Intracellular microelectrode and whole-cell patch-clamp recordings were used to investigate a Ba(2+)-induced regenerative depolarization and its underlying Ba2+ current in the ciliary body epithelial cells of the rabbit eye. Exposure of these epithelial cells to 4-10 mmol l-1 Ba2+ depolarized the membrane potential and caused the generation of one or more spikes, before the membrane potential reached a steady-state level. The spikes, but not the slow phase of depolarization, could be blocked with Co2+ (2 mmol l-1), Gd3+ (25 mumol l-1), La3+ (20 mumol l-1), Cd2+ (10 mumol l-1), verapamil (30 mumol l-1) and nifedipine (1 mumol l-1). Tetrodotoxin at 100 nmol l-1 had no effect. In the absence of Na+, but in the presence of external Ba2+, step depolarization of the membrane potential activated an inward current that could be blocked with Co2+ (2 mmol l-1), Cd2+ (10 mumol l-1) and nifedipine (1 mumol l-1), but not with Ni2+ (50 mumol l-1) or omega-conotoxin (1-10 mumol). This inward current could be enhanced with the dihydropyridine agonist (+/-)BAY K 8644 (1 mumol l-1). The inactivation characteristics of the inward current (v1/2 = -38.7 mV, k = 12.6 mV) is most like that seen in neurons. These findings indicate that the epithelial cells of the ciliary body possess dihydropyridine-sensitive, voltage-activated Ca2+ channels.

Action Potentials↗

PubMed1994

Ligand-gated currents of alpha and beta ganglion cells in the cat retinal slice.

1. We studied the receptor pharmacology of the ligand-gated currents of ON- and OFF- alpha and beta ganglion cells in a cat retinal slice preparation using the whole cell recording variation of the patch-clamp technique. Cat retinal slices were cut in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer and incubated in a bicarbonate-buffered solution. Ganglion cells were voltage clamped at -70 mV in HEPES-buffered Ringer solution. The pipette solution contained a low concentration of Cl- to distinguish mixed cationic from Cl(-)-mediated conductances, and Lucifer yellow (0.5%) was included for identification of the cell type. 2. In Ringer solution containing 1.2 mM Mg2+, current-voltage (I-V) curves of responses to the excitatory amino acid agonist (EAA) N-methyl-D-aspartate (NMDA) (200 microM) revealed a J-shaped function. In Mg(2+)-free Ringer solution containing 200 microM Cd2+ to block synaptic transmission, NMDA (200 microM) elicited an inward current 5-8 times larger at -70 mV. In both conditions I-V curves of the NMDA-induced currents reversed near 0 mV. These results suggest that there are NMDA EAA receptors present directly on the dendrites of alpha and beta ganglion cells. Responses to NMDA were blocked by +/- 2-amino-7-phosphonoheptanoic acid (AP7) (200 microM). 3. In Ringer solution containing 200-1,000 microM Cd2+ to block synaptic transmission, both ON- and OFF- alpha and beta cells responded to kainic acid (10-50 microM), alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA) (20-70 microM), and quisqualic acid (0.1-30 microM) with inward currents that reversed near 0 mV. These responses were blocked by the quinoxaline EAA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 microM). The metabotropic agonists 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) (25 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) and L-2-amino-4-phosphonobutyric acid (L-APB) (50 microM) in the presence of Cd2+ evoked little or no response for all cells tested. 4. In the presence of Cd2+, alpha and beta cells responded to gamma-amino-butyric acid (GABA) (200 microM) and glycine (200 microM) with inward currents that reversed near -35 mV, the calculated chloride equilibrium potential Ecl. Responses to GABA and glycine were both strongly desensitizing. (+)Bicuculline methyl chloride (20 microM) blocked an average of 90% of the inward current evoked by 200 microM GABA on all ganglion cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Photoreceptor degeneration in vitamin A deprivation and retinitis pigmentosa: the equivalent light hypothesis.

Long-term exposure of the retina to constant illumination is known to produce irreversible degeneration of photoreceptors. We propose that similar mechanisms may be involved in photoreceptor degeneration produced by vitamin A deprivation and some forms of retinitis pigmentosa (RP). Evidence is reviewed suggesting that the free opsin present during vitamin A deprivation or the mutated opsin present in some forms of RP excite the visual transduction cascade. This would produce a constant 'equivalent light' that triggers photoreceptor degeneration. Continuous real or equivalent light may produce outer segment degeneration by interfering with circadian processes, such as protein synthesis and disc shedding and lead to the loss of photoreceptors including those not expressing the mutant gene.

Animals↗

PubMed1993

Tissue culture of rabbit ciliary body epithelial cells on permeable supports.

The aqueous humor is produced by the epithelium of the ciliary body, a complex structure encircling the anterior segment of the eye. Aqueous humor production occurs by active transport, but the mechanism of this process is not understood. To produce a preparation in which active transport can be investigated, we have attempted to prepare cultures suitable for measurements of ion and water flux. We have grown rabbit ciliary body epithelial cells on permeable supports, coated with several extracellular matrix proteins. We then examined the ability of these proteins to promote the growth of a differentiated layer of epithelial cells. Non-pigmented and pigmented cells formed sheets of contiguous cells when grown on a variety of support media. The most successful substrate was a permeable support produced by Falcon/Becton Dickinson coated with a mixture of collagen IV, laminin and heparan sulfate. Under these conditions, cultures could be maintained for several months, but pigmented cell cultures did not develop a measurable transepithelial resistance (TER), and the TER of non-pigmented cell cultures was typically only 20-30 omega cm2. Much higher TERs exceeding 200 omega cm2 could be measured from non-pigmented cell cultures 3-5 days after plating, but these high values were unstable. Examination of the cultures with electron microscopy revealed that the cells were partially differentiated with the formation of a basal lamina and intercellular junctions. Labelling with a specific monoclonal antibody marker for tight junction protein (ZO-1) suggested that non-pigmented cell cultures showed extensive tight junction formation. The low TER of the non-pigmented cell cultures appears therefore not to be due to the lack of tight junctions but rather to the presence of spaces between cells.

Animals↗

PubMed1993

The excitatory and inhibitory amino acid receptors on horizontal cells isolated from the white perch retina.

1. The distribution and the properties of receptors to the inhibitory amino acid glycine (GLY) and the excitatory amino acid glutamate (GLU) and its analogues kainate (KA), quisqualate (QUIS), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and N-methyl-D-aspartate (NMDA), were studied with whole-cell and outside-out patch-clamp techniques on all four types of horizontal cells isolated from the retina of white perch. 2. Glycine at concentrations above 30 microM evoked whole-cell current responses from two types of horizontal cells (H2 and H4). The other two types of horizontal cells (H1 and H3) were unresponsive to GLY (30 microM-3 mM). 3. Responses elicited by GLY from H2 and H4 cells were similar, consisting of inward currents that desensitized with a half-decay time of 0.5-2 s at glycine concentrations between 100 and 500 microM. GLY-activated currents were inhibited by the glycine receptor antagonist strychnine (STRYCH). Current responses evoked by GLY reversed at the Cl- equilibrium potential. 4. Dose-response analysis of peak currents induced by GLY revealed a Hill coefficient of 2.0 +/- 0.1 (mean +/- SD, n = 3) and an median effective concentration (EC50) of 85 +/- 2 microM (n = 3). 5. Single glycine receptor channels recorded from outside-out patches had a main-state conductance of 47 +/- 4 pS (n = 3). 6. Every type of horizontal cell from the white perch responded to GLU, KA, QUIS, and AMPA but none responded to exogenously applied NMDA (200 microM) or NMDA (200 microM) + GLY (1 microM) in a Mg+2-free bathing solution. 7. The ratio of the amplitude of responses to GLU, KA, QUIS, and AMPA remained nearly constant among all the horizontal cells tested, suggesting there might be only a single population of non-NMDA receptors on these cells. 8. QUIS and KA both elicited responses from the horizontal cells. When applied together with KA, QUIS competitively antagonized the responses of horizontal cells to KA. 9. The results demonstrated the existence of an inhomogeneous distribution of strychnine-sensitive glycine receptors and a homogeneous distribution of non-NMDA type glutamate receptors among the four types of white perch horizontal cells.

Amino Acids↗