Nitric oxide donor stimulated increase of cyclic GMP in the goldfish retina.

Nitric oxide (NO) activates soluble guanylyl cyclase (sGC) and the resulting increase in cyclic guanosine monophosphate (cGMP) is an important intracellular signalling pathway in the vertebrate retina. Immunocytochemical detection of cGMP following exposure to NO donors has proven an effective method of identifying cells that express sGC. While such an approach has proven useful for the study of several vertebrate retinas, it has not been applied to the well-characterized teleost retina. Therefore, in the present study, we have applied this approach to the retina of the goldfish (Carassius auratus). In the presence of the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), incubation of goldfish eyecups in Ringer's solution containing (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) increased cGMP-like immunoreactivity (cG-ir) in bipolar, horizontal, amacrine, and ganglion cells and in ganglion cell axons and optic nerve. Weak labeling was observed in horizontal cells but no change in cG-ir was noted within photoreceptors. The NO donor-stimulated increases of cG-ir in horizontal, bipolar, amacrine, and ganglion cells are consistent with known physiological effects of NO on these neurons. The physiological significance of NO action at the level of optic nerve is not known. The lack of an effect of SNAP on cG-ir in photoreceptors was unexpected, as there are known physiological actions of NO, mediated by cGMP, on these neurons. Although this may be due to insufficient sensitivity of immunolabeling, this result may indicate a difference between isoforms of sGC or cGMP PDE in these neurons, compared to neurons where exogenous NO increased cG-ir.

Suppressive actions of betaxolol on ionic currents in retinal ganglion cells may explain its neuroprotective effects.

Betaxolol, a beta 1-selective adrenoceptor antagonist, is widely used in the treatment of glaucoma. In addition to its ocular hypotensive effects, betaxolol has been suggested to act as a retinal neuroprotective agent (Osborne et al., 1997). To investigate possible mechanisms underlying the neuroprotective effects, we tested the actions of betaxolol on ion channels and calcium signaling in isolated retinal ganglion cells. Betaxolol (50 microM) reduced by about 20% the high-voltage-activated (HVA) Ca channel currents in ganglion cells isolated from tiger salamander retina. In contrast, the beta 1-adrenoceptor antagonists propranolol (10 microM) and timolol (50 microM) had no inhibitory actions on HVA Ca channel currents. The L-type Ca channel antagonist, nisoldipine, blocked the HVA Ca channel current partially and the remaining current was not inhibited by betaxolol. Outward current was inhibited in the presence of betaxolol. Both iberiotoxin (IBTX; 10 nM), a selective inhibitor of large-conductance Ca-activated K channels, and Cd2+ (100 microM), which suppresses Ca-activated K channels subsequent to its block of Ca channels, reduced outward current and the remaining current was not blocked significantly with betaxolol. In the presence of betaxolol, Na channel currents were reduced by about 20%, as were currents evoked by glutamate (10 mM) and GABA (1 mM). Current clamp recordings from isolated ganglion cells showed that betaxolol had several effects on excitability: spike height decreased, repetitive spike activity was suppressed, spike width increased and hyperpolarization following spikes was reduced. Calcium imaging in isolated rat retinal ganglion cells revealed that betaxolol inhibited glutamate-induced increases in [Ca2+]i. These results suggest that betaxolol has a diversity of suppressive actions on ganglion cell ion channels and that, as a consequence, one of the net actions of the drug is to reduce Ca2+ influx. The subsequent reduction in [Ca2+]i may contribute to the apparent neuroprotective actions of betaxolol in promoting ganglion cell survival following ischemic insult, as may occur in glaucoma and retinal disease.

Taurine blocks spontaneous cone contraction but not horizontal cell dark suppression in isolated goldfish retina.

The objective of this study was to investigate the effects of taurine on cone retinomotor movements and the responses of cone-driven horizontal cells in dark-adapted teleost retina. In isolated goldfish retina preparations maintained in the dark, cones spontaneously contracted, and the responses of horizontal cells were suppressed. Addition of 5 mM taurine to the physiological solution blocked the spontaneous contraction of cones in the dark but did not block the dark-suppression of horizontal cell responses. These results indicate that the mechanism that leads to horizontal cell dark suppression is not sensitive to taurine. Although both cone retinomotor position and horizontal cell responsiveness are known to be modulated by dopamine, the present results do not support the hypothesis that taurine inhibits dopamine release in the dark because only spontaneous cone contraction was affected by taurine. These results also indicate that spontaneous cone contraction in the dark is not the cause of horizontal cell dark suppression because, in the presence of taurine, cones were elongated yet horizontal cell responses were still suppressed. Consequently, these results make it clear that horizontal cell dark suppression is not an artifact produced by incubating isolated teleost retina preparations in taurine-free physiological solution.

Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells.

1. We used whole-cell patch clamp and fluorescent calcium imaging techniques to investigate the effects of adenosine 5'-triphosphate (ATP) on membrane currents and intracellular calcium concentration ([Ca2+]i)in rat retinal pigment epithelial (RPE) cells. In 62 % of RPE cells, application of 100 microM ATP elicited a fast inward current at negative membrane potentials. In 38 % of RPE cells recorded, a biphasic response to ATP was observed in which activation of the fast inward current was followed by activation of a delayed outward current. 2. The ATP-activated inward current was a non-selective cation (NSC) current that showed inward rectification, reversed at -1.5 +/- 1 mV and was permeable to monovalent cations. The NSC current was insensitive to the P2 purinoceptor antagonists, suramin or PPADS but was activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 3. The outward current activated by ATP reversed at -68 +/- 3 mV (equilibrium potential for potassium (EK) = -84 mV) and was blocked by Ba2+ ions, consistent with the activation of a K+ conductance. The outward K+ conductance was also reduced by the maxi-KCa channel blocker iberiotoxin (IbTX; 10 nM), suggesting that ATP activated an outward Ca2+-activated K+ channel in rat RPE cells. The Ca2+-activated K+ current (IK(Ca)) was also activated by the purinoceptor agonists UTP, ADP and 2MeSATP. 4. In fluo-3 or fluo-4 loaded RPE cells, ATP and the pyrimidine agonist UTP elevated [Ca2+]i. The increase in Ca2+ was not dependent on extracellular Ca2+ influx, but was sensitive to the Ca2+-ATPase inhibitor thapsigargin, confirming the involvement of intracellular Ca2+ stores release. 5. These results suggest that rat RPE cells express both P2X purinoceptors that gate activation of a non-selective cation conductance and G protein-coupled P2Y purinoceptors that mediate Ca2+ release from intracellular stores and activation of a calcium-activated K+ current.

The modulation of intercellular coupling in the retina.

Gap-junction coupling between retinal neurons of the same (homologous) or different (heterologous) type can be modulated dynamically by neurotransmitters and by the level of ambient illumination. The homologous coupling of both horizontal cells and rod (AII) amacrine cells is reduced by dopamine, but there appear to be cell and species differences in how the ambient illumination interacts with the dopaminergic system to modulate the coupling. Both the homologous coupling of horizontal cells and the heterologous coupling between AII amacrine cells and cone bipolar cells are reduced by nitric oxide, but it is not known if changes in illumination modulate coupling through an endogenous nitrergic mechanism. The heterologous coupling between rod and cone photoreceptors is modulated by illumination, but the endogenous mechanism has not been identified.

Calcium-sensitive calcium influx in photoreceptor inner segments.

The effect of external calcium concentration ([Ca2+]o) on membrane potential-dependent calcium signals in isolated tiger salamander rod and cone photoreceptor inner segments was investigated with patch-clamp and calcium imaging techniques. Mild depolarizations led to increases in intracellular Ca2+ levels ([Ca2+]i) that were smaller when [Ca2+]o was elevated to 10 mM than when it was 3 mM, even though maximum Ca2+ conductance increased 30% with the increase in [Ca2+]o. When external calcium was lowered to 1 mM [Ca2+]o, maximum Ca2+ conductance was reduced, as expected, but the mild depolarization-induced increase in [Ca2+]i was larger than in 3 mM [Ca2+]o. In contrast, when photoreceptors were strongly depolarized, the increase in [Ca2+]i was less when [Ca2+]o was reduced. An explanation for these observations comes from an assessment of Ca2+ channel gating in voltage-clamped photoreceptors under changing conditions of [Ca2+]o. Although Ca2+ conductance increased with increasing [Ca2+]o, surface charge effects dictated large shifts in the voltage dependence of Ca2+ channel gating. Relative to the control condition (3 mM [Ca2+]o), 10 mM [Ca2+]o shifted Ca2+ channel activation 8 mV positive, reducing channel open probability over a broad range of potentials. Reducing [Ca2+]o to 1 mM reduced Ca2+ conductance but shifted Ca2+ channel activation negative by 6 mV. Thus the intracellular calcium signals reflect a balance between competing changes in gating and permeation of Ca2+ channels mediated by [Ca2+]o. In mildly depolarized cells, the [Ca2+]o-induced changes in Ca2+ channel activation proved stronger than the [Ca2+]o-induced changes in conductance. In response to the larger depolarizations caused by 80 mM [K+]o, the opposite is true, with conductance changes dominating the effects on channel activation.

Modulation of endogenous dopamine release in the fish retina by light and prolonged darkness.

The effect of light stimuli and prolonged darkness on the release of endogenous dopamine was measured in the white perch and hybrid bass retinas. Isolated retinas were superfused and released dopamine was measured using extraction and high-pressure liquid chromatography separation techniques. Potassium-induced release did not depend on the background illumination nor on the period of previous darkness. Steady white light did not affect release, but flickering light of 2 Hz increased release about two-fold. During prolonged darkness, the release of dopamine increased steadily over the test period of 2 h, but only if the experiments were performed at night. During the day such an increase was not observed. The increased release during prolonged darkness at night was turned off by a short period of steady white light. The release patterns obtained from the white perch and the hybrid bass were similar. However, the hybrid bass retina showed much lower levels of dopamine than did the white perch retina.

Optical recordings of the effects of cholinergic ligands on neurons in the ganglion cell layer of mammalian retina.

Cholinergic regulation of the activity of rabbit retinal ganglion cells and displaced amacrine cells was investigated using optical recording of changes in intracellular free calcium ([Ca2+]i). Labeling of neurons in the mature retina was achieved by injecting calcium green-1 dextran (CaGD) into the isolated retina. Nicotine increased ganglion cell [Ca2+]i, affecting every loaded cell in some preparations; the pharmacology of nicotine was consistent with an action at neuronal nicotinic receptors, and specifically it was kappa-(neuronal-)bungarotoxin-sensitive but alpha-bungarotoxin-insensitive. Muscarine also raised [Ca2+]i, but it was less potent than nicotine, affecting only a subpopulation of ganglion cells, with an M1-like muscarinic receptor pharmacology. Neither the nicotine- nor muscarine-induced increases of ganglion cell [Ca2+]i were blocked by the glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and aminophosphonopentanoic acid. Therefore, the effects of cholinergic agonists on ganglion cell [Ca2+]i were not attributable to an indirect effect mediated by glutamatergic bipolar cells. The effects of nicotine and muscarine were abolished in calcium-free solution, indicating that the responses depend on calcium influx. Displaced (Cb) cholinergic amacrine cells were also loaded with CaGD and were identified by selective labeling with the nuclear dye 4',6-diamidino-2-phenyl-indole. Cb amacrine cells did not respond to either nicotine or muscarine, but responded vigorously to the glutamate receptor agonist kainic acid. There is anatomical evidence indicating that cholinergic amacrine cells make synaptic contact with each other, but the present results do not support the hypothesis that communication between these cells is cholinergic.

Dark-suppression and light-sensitization of horizontal cell responses in the hybrid bass retina.

The responsiveness of luminosity-type horizontal cells, recorded intracellularly from isolated hybrid bass retinas, decreased after superfusion for 2 h in constant darkness. Responsiveness was subsequently increased (light-sensitized) up to 10-fold after exposure to several short (approximately 0.5 min) periods of continuous illumination. The increase in horizontal cell responsiveness following light-sensitization was due to an increase of peak response amplitude rather than a reduction of peak response time. The increased responsiveness after light-sensitization was intensity-dependent with brighter sensitizing stimuli causing a greater increase than dimmer stimuli. The extent of LHC dark-suppression was affected by the time of day, being greater when induced during the night than during the day. However, there was no significant difference in horizontal cell responsiveness after light-sensitization in retinas studied during the night compared to those studied during the day. The responsiveness of light-sensitized horizontal cells from isolated hybrid bass retinas was found to be suppressed by relatively brief periods of darkness. The responsiveness of horizontal cells, that were first light-sensitized, decreased by more than 50% following only 5 min of darkness. Suppression of light-sensitized horizontal cell responsiveness after such a short time in the dark has not been described in other teleost retinas. The suppression of light-sensitized horizontal cell responsiveness in hybrid bass retinas may be rapid in comparison to other teleosts.

Threshold and chromatic sensitivity changes in fish cone horizontal cells following prolonged darkness.

The light-evoked responses of L-type cone horizontal cells in the teleost retina were studied following a prolonged period of complete darkness. Intact, isolated white perch retinas were superfused in complete darkness for more than 90 min, following which horizontal cells were impaled without the aid of any light flashes. Following this prolonged darkness, L-type cone horizontal cell light responses to dim and bright full-field stimuli were slow and small in amplitude and response duration to bright stimuli was considerably longer than stimulus duration. In addition, absolute threshold was 2 log units lower than typical for cone horizontal cells and spectral sensitivity to shorter wavelengths was increased. Following bright light stimulation, light responses became more transient and increased in amplitude, reaching 40-50 mV to bright flashes. Moreover, absolute threshold increased and responses to spectral stimuli were similar to those observed typically for L-type cone horizontal cells after light-sensitization. These results suggest that following prolonged darkness, cone input to cone horizontal cells is reduced and rod input is present.

Evidence for D2 receptor regulation of dopamine release in the goldfish retina.

The possible existence of a dopamine D2 receptor-mediated regulation of dopamine release was investigated in the goldfish retina. Isolated retinas were preloaded with [3H]dopamine and superfused with D2 dopamine receptor agonists or antagonists to determine if there was an effect on [3H]dopamine release. The D2 receptor antagonist sulpiride increased both baseline [3H]-dopamine release and [3H]dopamine release induced by an increase in extracellular potassium concentration. The D2 receptor agonists LY-171555 and RU-24213 did not reduce baseline [3H]dopamine release but completely inhibited [3H]dopamine release induced by an increase in [K+]o. This action of the D2 agonists was blocked by sulpiride. These studies demonstrate the existence of D2 receptor, possibly autoreceptor, regulation of dopamine release in the teleost retina.

Effects of norepinephrine on [3H]dopamine release and horizontal cell receptive-field size in the goldfish retina.

Norepinephrine increased the release of pre-loaded [3H]dopamine from goldfish retinas. Pharmacological studies suggested that the norepinephrine-induced [3H]dopamine release was due to an exchange mechanism between norepinephrine and pre-loaded [3H]dopamine. Norepinephrine also depolarized and reduced the receptive-field size of horizontal cells in goldfish retinas. The action of norepinephrine on horizontal cells was probably not due to the release of endogenous dopamine because the effect of norepinephrine was not abolished in retinas in which all dopaminergic neurons had been destroyed by prior treatment with 6-hydroxydopamine. The pharmacology of the effect of norepinephrine on horizontal cells suggested that it was due to an agonist action of norepinephrine acting at horizontal cell dopamine receptors. It is still unclear whether endogenous norepinephrine is a regulator of dopamine release in the fish retina. Consequently, the function of the putative norepinephrine-containing amacrine cells of the fish retina remains to be elucidated.

A new type of interplexiform cell in the goldfish retina is PNMT-immunoreactive.

A new cell type immunoreactive for phenylethanolamine N-methyltransferase, the synthesizing enzyme for epinephrine, has been identified in the goldfish retina. The somata of the immunoreactive cells were located in the proximal inner nuclear layer and immunoreactive processes were located in both the inner and outer plexiform layers, suggesting that this cell may be an interplexiform cell. Confocal microscopy was used to establish that the putative adrenergic neurites in the outer plexiform layer were located between the somata of horizontal cells and photoreceptor cell synaptic terminals. Intracellular recordings from horizontal cells demonstrated that epinephrine had an effect on horizontal cells, but that the action of epinephrine was consistent with an effect on dopamine receptors, rather than adrenergic receptors. The function of the putative 'I3' interplexiform cell therefore remains unclear, possibly modulating horizontal cell function with a transmitter other than epinephrine or affecting photoreceptors or the retinal pigment epithelium rather than horizontal cells.

Neuromodulation of pigment movement in the RPE of normal and 6-OHDA-lesioned goldfish retinas.

The role of dopamine as the endogenous signal-initiating light-dependent changes in the distribution of pigment granules in goldfish retinal pigment epithelium was investigated. In normal retinas, light adaptation resulted in the dispersion of pigment granules. This effect of light was mimicked by the intraocular injection of dopamine or serotonin, which is thought to increase endogenous dopamine release, into dark-adapted eyes. The effect of light, dopamine, or serotonin on dark-adapted retinas was blocked by the dopamine receptor antagonists haloperidol and sulpiride. However, lesioning the endogenous source of retinal dopamine, by prior intraocular injection of 6-hydroxydopamine (6-OHDA), did not block the dispersion of pigment granules in light-adapted retinas. No significant differences in pigment dispersion were noted between unlesioned and lesioned light- or dark-adapted retinas. However, the effect of light on pigment dispersion was no longer blocked by haloperidol or sulpiride in 6-OHDA lesioned animals. Dopamine and serotonin mimicked the effect of light when injected into lesioned dark-adapted eyes, but their effects were also not blocked by haloperidol or sulpiride. These results suggest that dopamine, acting on D2 receptors, is sufficient to induce pigment migration in unlesioned animals. In 6-OHDA-lesioned animals, however, pigment migration is mediated by a receptor mechanism other than D2.

Background illumination reduces horizontal cell receptive-field size in both normal and 6-hydroxydopamine-lesioned goldfish retinas.

The effect of background illumination on horizontal cell receptive-field size and dye coupling was investigated in isolated superfused goldfish retinas. Background illumination reduced both horizontal cell receptive-field size and dye coupling. The effect of light on horizontal cell receptive-field size was mimicked by treating the retina with 20 microM dopamine. To test the hypothesis that the effects of light were due to endogenous dopamine release, the effect of light was studied in goldfish retinas in which dopaminergic interplexiform cells were lesioned using 6-hydroxydopamine treatment. In lesioned retinas, background illumination reduced both horizontal cell receptive-field size and dye coupling. Furthermore, the effect of background illumination on unlesioned animals could not be blocked by prior treatment with the D1 dopamine receptor antagonist SCH-23390. These results suggest that, in goldfish retina, dopamine release is not the only mechanism by which horizontal cell receptive-field size could be reduced by light.

Gap junction particle density of horizontal cells in goldfish retinas lesioned with 6-OHDA.

The I1 dopaminergic interplexiform cells of the fish retina are believed to modulate horizontal cell coupling by increasing gap junction resistance. Dopamine also modulates the morphology of horizontal cell gap junctions and mimics the effects of light adaptation. To determine whether the light-dependent changes in gap junction morphology are due to endogenous dopamine release, horizontal cell gap junctions were studied in goldfish retinas lacking dopaminergic neurons. Dopaminergic interplexiform cells were destroyed by intraocular injections of 6-hydroxydopamine in both eyes. After lesioning, fish were treated in one of four ways: (1) light-adapted, (2) dark-adapted (1 hour), (3) light-adapted and given an intraocular injection of dopamine, or (4) dark-adapted (1 hour) and injected with dopamine. The effectiveness of lesioning was evaluated by autoradiographic detection of [3H]-dopamine uptake in the retina of one eye. Retinas in which lesioning of the contralateral eye was deemed effective were processed for freeze-fracture electron microscopy and the particle density of horizontal cell gap junctions determined. Lesioned retinas, whether light- or dark-adapted, had elevated horizontal cell soma gap junction particle densities compared to lesioned retinas treated with dopamine. These results demonstrate that high soma gap junction particle densities can be correlated with the absence of dopamine and low densities associated with the presence of dopamine. The differences in gap junction particle density between lesioned and lesioned + dopamine-treatment were similar to differences between nonlesioned dark-adapted (1 hour) and light-adapted retinas, respectively. Therefore, the particle density of light- and dark-adapted soma gap junctions suggests a greater release of dopamine in light-adapted fish than in 1 hour dark-adapted fish.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopaminergic regulation of horizontal cell gap junction particle density in goldfish retina.

Light- or dark-adapted goldfish (Carassius auratus) retinas were treated with dopamine, which is believed to uncouple horizontal cells via D1 receptors, or with the dopamine antagonist haloperidol. Aldehyde-fixed retinas were freeze-fractured and the replicas examined by electron microscopy to identify horizontal gap junctions. The density (number per micron2) of intra-membrane particles of horizontal cell soma gap junctions was significantly lower in light-adapted and dopamine-treated retinas than in dark-adapted and haloperidol-treated retinas. There was no statistically significant difference between gap junction particles densities in (I) light-adapted (untreated) and in dopamine-treated (light- or dark-adapted) retinas, or between (II) dark-adapted (untreated) and haloperidol-treated (light- or dark-adapted). These results suggest that the uncoupling of horizontal cell somas by dopamine is accompanied by a decrease in gap junction particle density and that there is a greater release of dopamine during light-adaptation than dark-adaptation. Unlike horizontal cell somas, horizontal cell axon terminals did not show consistent changes in gap junction particle density with light- or dark-adaptation. Although the data suggests that there may be a reduction in axon terminal gap junction particle density with dopamine treatment, this effect is not reversible with haloperidol treatment. Our results suggest that the regulation of gap junctions may differ at two sites within the same cell.

The tight junction as a barrier to cholesterol in canine epithelial cells.

Filipin has been used to test several models of continuity or flow of lipid components through the tight junction. Cultured canine kidney cells (MDCK) were fixed and incubated in the presence of filipin. Freeze-fracture replicas were analyzed and densities of filipin-cholesterol complexes measured. Fractures of membranes linked with tight junctions were compared statistically to determine whether filipin-cholesterol complexes (protrusions and pits, independently) were randomly distributed between the two membranes of cells separated by the tight junction. The results indicate that filipin-cholesterol complexes are not randomly distributed across the tight junction. If the density of filipin-cholesterol complexes is an accurate indication of membrane cholesterol concentration, then there is a difference in the cholesterol concentration between leaflets of membranes joined by tight junctions and models of the tight junction which suggest leaflet continuity across the junction are in error.