Karl-Friedrich Schmidt

Sodium nitroprusside alters dark voltage and light responses in isolated retinal rods during whole-cell recording.

Dark voltage and light responses of isolated retinal rods of Rana esculenta were investigated by employing the whole-cell patch-clamp technique. When the recording pipette was filled with a medium devoid of nucleotides, a spontaneous hyperpolarization of the dark voltage partly due to a diffusional loss of cGMP and its precursor GTP and a retardation in the recovery of the light responses was observed. The larger part of the retardation of the light responses was prevented by 1 mM ATP. Addition of GTP attenuated the hyperpolarization, but did not abolish it completely. When the nitric-oxide-releasing substance sodium nitroprusside plus GTP was applied, the tendency of hyperpolarization disappeared and a stable dark voltage or even a slight depolarization was measured during the whole-cell recording period. Similar results were also obtained when GTP was given in combination with either EGTA or IBMX which are both known to interfere with the cGMP regulating enzymes in retinal rods. In addition to its effects on the dark voltage, an acceleration of the recovery phase of the light responses by sodium nitroprusside was also observed. Our observations strongly suggest that sodium nitroprusside activates guanylate cyclase in photoreceptors, as it does in other tissues, but we cannot exclude with certainty an effect on the phosphodiesterase.

Studies on the dopamine-dependent modulation of amino acid-gated currents in cone horizontal cells of the perch (Perca fluviatilis)

The whole-cell patch-clamp technique was employed to record membrane currents from isolated horizontal cells in culture. Concentration-response relationships for currents induced by L-glutamate, kainate and quisqualate were measured. Preincubation with dopamine changed the parameters of the concentration-response curves in a particular way for each agonist. The maximum currents induced by glutamate increased by 50-100% and the EC50-values were slightly shifted to higher values. The increase of kainate-induced maximum currents after dopamine incubation did not exceed 30% but the EC50-values were clearly shifted to lower concentrations. Quisqualate-induced maximum currents were not enhanced by dopamine preincubation but the EC50-values were shifted to lower concentrations. The dopamine-dependent modulation was affected by removal of magnesium and preincubation with concanavalin A and aniracetam. The concentration-response relation and the time-course of the dopamine effect on glutamate-induced currents is described.

Divalent cations modulate glutamate receptors in retinal horizontal cells of the perch (Perca fluviatilis)

Divalent cations had two effects on concentration-response relations of glutamate induced membrane currents recorded from retinal horizontal cells. The first effect was a reduction of maximum currents. Barium, magnesium, cobalt, nickel and an increased calcium concentration caused reductions of maximum currents between 14% and 70%. The second effect of divalent cations was related to the dopamine dependent modulation of glutamate receptors in horizontal cells. The dopamine dependent enhancement of glutamate gated currents requires the presence of divalent cations besides calcium in the extracellular solution. Without such divalent cations application of dopamine caused no increase of the maximum currents induced by glutamate, and only a slight shift of the half maximal saturation concentration was observed. Addition of magnesium or barium cations in millimolar concentration was sufficient to completely restore the dopamine dependent modulation.

Effect of guanine nucleotides on the dark voltage of single frog rods

Single frog rods consisting of the outer segment and the ellipsoid were investigated by the whole-cell patch-clamp technique. When the recording pipette was filled with a simple intracellular medium containing potassium as the principal cation, a slow increase in dark voltage (hyperpolarization) associated with a decay of the photoresponses was observed. The hyperpolarization started at a dark voltage of -27 +/- 8 mV, followed an exponential course, and leveled out at -52 +/- 6 mV. The time constant was proportional to the access resistance of the preparations. With a pipette medium containing a 0.5 or 1.0 microM cGMP, the initial dark voltage was shifted to more positive values and the tendency of hyperpolarization was clearly attenuated. Similar results were obtained with 1 mM GTP. The effects of GDP and of ATP were less significant. In experiments with 1 mM GTP plus 1 mM ATP, the dark voltage behaved as in experiments with only GTP. The stabilizing action of GTP was amplified by EGTA so that with 1 mM GTP plus 1 mM free EGTA the dark voltage was stable at a level of -15 mV. It is concluded that the preparations lose intracellular components such as cGMP and GTP by diffusion into the recording pipette and that the losses are prevented or reduced when the pipette medium contains these nucleotides in nearly physiological concentrations. For the internal transmitter cGMP, the results suggest that its free concentration does not exceed 1 microM.

Properties of glutamate-gated ion channels in horizontal cells of the perch retina

The effect of two different concentrations of L-glutamate and kainate on the gating kinetics of amino acid-sensitive non-NMDA channels were studied in cultured teleost retinal horizontal cells by single-channel recording and by noise analysis of whole-cell currents. When the glutamate agonist kainate was applied clearly parabolic mean-variance relations of whole-cell membrane currents (up to 3000 pA) indicated that this agonist was acting on one type of channels with a conductance of 5-10 pS. The cells were less sensitive when L-glutamate was used as the agonist and in most cases whole-cell currents amounted to less than 200 pA. The mean-variance relation of glutamate induced currents was complex, indicating that more than one type of channel opening could be involved. Power spectra of whole-cell currents were fitted with two Lorentzians with time constants of approx. 1 and 5-20 msec. Effects on amplitudes and time constants of agonist concentrations are demonstrated. Two categories of unitary events with mean open times of approx. 1 and 7 msec and conductances of approx. 7 and 12 pS, respectively, were obtained in single-channel recordings from cell-attached patches at different concentrations of glutamate in the pipette.

Fibroblast growth factors alter light responses and dark voltage in retinal rods of the frog (Rana temporaria)

Fibroblast growth factors (FGF-1 and FGF-2) were applied intracellularly via whole-cell patch-clamp electrodes while the membrane voltage was recorded simultaneously. During recording the exchange of substances by diffusion between cytosol and pipette medium affects the cells function. Under control conditions, the loss of nucleotides is reflected by a slow hyperpolarization of the dark voltage and prolongated light responses. Addition of FGF-1 and FGF-2 to the pipette medium accelerated the time course of the hyperpolarization and intensified the prolongation of the light responses. The depolarization of photoreceptor cells after intracellular application of the nitric oxide (NO)-synthase cofactor nicotinamide adenine dinucleotide phosphate (NADPH) and the stabilization of light response recovery by L-arginine is abolished by FGF-2. FGF-2 was ineffective when it was applied together with the calcium chelator ethylene glycol-bis(2-aminoethylether)tetraacetate (EGTA). The results indicate a possible role of FGF in the regulation of NO and calcium in photoreceptor cells and may explain protective effects of FGF in degenerative processes of photoreceptor cells.

Effect of intracellularly applied sodium ions on the dark voltage of isolated retinal rods.

Isolated retinal rods of the frog consisting of the outer segment and the ellipsoid were patch-clamped and recorded in the whole-cell mode. The recording pipettes were filled with solutions of different composition in order to alter the cytoplasmic content of sodium, phosphate, and calcium ions, and guanine nucleotides. When a simple medium with potassium as the principal cation was used, the dark voltage slowly approached more negative values. This tendency of spontaneous hyperpolarization was reduced significantly when cGMP or GTP were present in the pipette medium. Sodium ions, on the other hand, clearly increased the speed of hyperpolarization. In the presence of sodium (20 mM), the stabilizing effect of GTP did not occur and that of cGMP was clearly diminished. Phosphate (20 mM) neutralized the sodium effect. High calcium levels (100 microM) did not measurably influence the time course of hyperpolarization. We conclude that the normal cytoplasmic sodium level in rods does not exceed 10 mM and that higher internal sodium concentrations interfere with the sodium-calcium exchange mechanism.

Configuration of light responses in isolated retinal rods

The whole-cell patchclamp technique was employed to investigate the light responses of single retinal rods of the frog (Rana esculenta and R. temporaria). In the majority of experiments, completely isolated cells were studied. Coupling with neighboring cells gave rise to a more complex response configuration. Responses were recorded under voltage-clamp and under current-clamp conditions. Stimulus response curves were measured in experiments with local stimuli illuminating only parts of the outer segment. Metabolic factors such as cGMP, GTP and ATP were also tested and were found to have specific and different influences on the response configurations. When the recording pipette was filled with an intracellular medium devoid of nucleotides, a retardation in the recovery of the light responses was observed during the course of an experiment. Addition of 1 mM ATP to the pipette medium prevented the larger part of the retardation, while 1 mM GTP accelerated the response recovery at the beginning of an experiment but did not prevent a subsequent retardation. Micromolar concentrations of cGMP were sufficient to elicit both a depolarization of the photoreceptor membrane and an increase in the response duration. These results show that, in single photoreceptors, the configuration of light responses not only depends on the stimulus parameters but also on those properties of the cells that are directly controlled by their nucleotide metabolism.

Protein kinase C does not mediate the dopamine-dependent modulation of glutamate receptors in retinal horizontal cells of the perch (Perca fluviatilis)

The whole-cell patch-clamp technique was employed to record membrane currents from cultured horizontal cells of the perch (Perca fluviatilis). The cells were voltage clamped and slowly superfused with an extracellular solution containing L-glutamate. The glutamate concentration in the bath was continuously measured with the help of photodiode and a dye which accumulated in the bath together with the agonist. The PKA-activator forskolin mimicked the effect of dopamine and enhanced glutamate-induced currents, while application of the PKC stimulator PMA or the synthetic diacylglycerol analogue OAG had no significant effects on the dose-response curves of glutamate induced-currents. These results may indicate that the modulation of glutamate receptors in fish horizontal cells is not mediated via a PKC-dependent pathway.