Ecology. A true-blue vision for the Danube.
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Animals↗
Biomedical subjects
K F Schmidt
Publications and source records attributed to K F Schmidt.
Ecology. Restoring the vitality of rich wetlands.
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Animals↗
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.
Animals↗
Measurement of concentration-response relationships by concentration-ramp application of agonists.
Dose-response relationships of agonist- induced membrane currents were obtained during accumulation of agonist and a simultaneously applied dye in the culture dish. The concentration of the dye was continuously measured using a laboratory-made photometer. When the superfusate was switched from the control solution to a test solution containing an agonist and an inert dye (e.g. Fast Green) both accumulated in the bath together and the change of dye concentration altered the intensity of light falling through the objective. The change in light intensity in an area directly surrounding the cell under study was measured by the photometer. The normal microscope lamp was used as the light source and a colour filter was placed between the microscope lamp and the condenser in order to enhance the signal-to-noise ratio of the photometer output. Assuming that the rates of accumulation and clearance of dye are similar to those of the agonist, it is possible to continuously measure the membrane current at given dye and agonist concentrations. To calculate the dose-response curves we calibrated the relationship between the dye concentration and the photometer voltage output.
Animals↗
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.
Animals↗
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.
Animals↗
Malaria research. Inbred parasites may spur resistance.
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Animals↗
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 cell's 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.
Amino Acid Oxidoreductases↗
Dopaminergic modulation of glutamate-activated channels in the central nervous system.
Modulation of glutamate-activated currents by dopamine was studied in identified central neurons, alpha-motoneurons of the chick and horizontal cells of the perch. This modulation is mediated by a cAMP-dependent protein phosphorylation. The activation and desensitization time constants of glutamate currents were determined before and after incubation with dopamine. In the horizontal cells ultrafast glutamate (AMPA or quisqualate) application prior to the the dopamine incubation gives rise to fast transient current responses which desensitize within less than 100 ms. Kainate produced higher steady state currents. After incubation of the cells with dopamine (100 nM) for 30s the desensitization was dramatically reduced, but the amplitudes of the steady state currents were similar to the transient control currents. Kainate activated currents were not affected. In alpha-motoneurons the exposure to dopamine (100 nM) for 1 min was sufficient to increase the peak and steady state amplitudes but not the desensitization time constant of glutamate activated currents. Here enhancement was specific to the kainate component of glutamate activated currents; the decreased variance of currents reflects increased kainate channel activation. Measurements of motoneuronal cAMP concentrations showed an increase following addition of dopamine. mRNA encoding both D1 and D2 dopamine receptor subtypes was detected. We conclude that the dopamine dependent modulation which is mediated by a protein phosphorylation is due to an alteration of the desensitization of AMPA type receptors in horizontal cells and of the activation of kainate type receptors in motoneurons.
Animals↗
Dopamine alters glutamate receptor desensitization in retinal horizontal cells of the perch (Perca fluviatilis).
The patch-clamp technique in combination with a fast liquid filament application system was used to study the effect of dopamine on the glutamate receptor desensitization in horizontal cells of the perch (Perca fluviatilis). Kinetics of ligand-gated ion channels in fish horizontal cells are modulated by dopamine. This modulation is presumably mediated by a cAMP-dependent protein phosphorylation. Before incubation with dopamine, the glutamate receptors of horizontal cells activate and desensitize with fast time constants. In the whole-cell recording mode, fast application of the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid prior to the dopamine incubation gives rise to fast transient currents with peak values of about 200 pA that desensitize within 100 ms. Kainate as agonist produced higher steady-state currents but no transient currents. After incubation of the cells with dopamine for 3 min, the desensitization was significantly reduced and the agonists L-glutamate, quisqualate, or alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid induced steady-state currents with amplitudes that were similar to the previously observed transient currents. Kainate-induced currents were only slightly affected. Fast desensitizing currents upon fast application of L-glutamate were also recorded from outside-out patches that were excised from horizontal cells before incubation with dopamine. The currents from excised patches desensitized to a steady-state level of about 0.2 of the peak amplitude with time constants of less than 2 ms. When the outside-out patches were excised from cells after dopamine incubation, steady-state currents were enhanced and no transient currents were observed. The results may indicate that the dopamine-dependent modulation of glutamate-induced currents, which is presumably mediated by a protein phosphorylation, is due to an alteration of the desensitization of the glutamate receptors.
Animals↗
Configuration of light responses in isolated retinal rods. A patch-clamp study.
The whole-cell patch-clamp 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.
Adenosine Triphosphate↗
Inhibition of nitric oxide synthase alters light responses and dark voltage of amphibian photoreceptors.
We studied the effects of competitive inhibitors of nitric oxide synthase (L-NMMA and L-NNA) on dark voltage and flash responses of retinal rods of the frog. Substances 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 cell's function. Under control conditions this exchange is reflected by a slow hyperpolarization of the dark voltage with time and a prolongated flash response recovery, which is mainly due to a loss of nucleotides. Application of L-NMMA and L-NNA accelerated the spontaneous hyperpolarization of the membrane voltage during the course of an experiment, while the recovery of the flash responses was slowed down. The effects observed upon intracellular application of NO-synthase inhibitors were opposite to those observed previously upon application of sodium nitroprusside. Sodium nitroprusside was much less effective when the intracellular calcium level was decreased by application of EGTA at the same time. It is reasonable to assume that the observed effects are linked to nitric oxide synthase and to a NO-dependent soluble guanylate cyclase. The results suggest that the activity of NO-synthase in photoreceptor cells has an influence on concentration and metabolic flux of cGMP in photoreceptors, which may be of relevance for flash response recovery and adaptation processes. It is likely that the regulation of the soluble guanylate cyclase requires a physiological level of calcium.
Amino Acid Oxidoreductases↗
L-arginine and nicotinamide adenine dinucleotide phosphate alter dark voltage and accelerate light response recovery in isolated retinal rods of the frog (Rana temporaria).
Effects of intracellularly applied L-arginine and nicotinamide adenine dinucleotide phosphate (NADPH) on the dark voltage and light responses of retinal rods were studied by means of the whole-cell patch-clamp technique. In this mode an exchange of substances by diffusion between cytosol and pipette medium occurs (Pflügers Arch., 411 (1988) 204-211). In retinal rods a loss of nucleotides is reflected by a hyperpolarization of the dark voltage and by a prolongation of the light responses (Vis. Neurosci., 2 (1989) 101-108). Intracellular application of L-arginine prevented the prolongation of the light responses and NADPH accelerated the light response recovery and in addition depolarized the photoreceptor cells. The effects were similar to those observed before upon application of the nitric oxide (NO)-releasing substance sodium nitroprusside (Vis. Neurosci., 9 (1992) 205-209). It is therefore assumed that the observed effects are linked to the NO-synthase and to an activation of a guanylate cyclase by NO. It is concluded that the level of NADPH in photoreceptor cells may affect the metabolic flux of guanosine 3',5'-cyclic monophosphate (cGMP).
Animals↗
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.
Animals↗
The troubling ghosts of scourges past.
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Communicable Disease Control↗
Size and chirality of intracellularly applied anions affect the function of isolated photoreceptors.
The effect of intracellularly applied anions on the function of retinal rods of the frog Rana esculenta was investigated by means of the whole-cell patch-clamp technique. When the recording pipette contained a medium based on potassium chloride, a slow spontaneous hyperpolarization was observed presumably due to a diffusional loss of the photoreceptor's internal transmitter cyclic guanosine monophosphate (cGMP) and its precursor guanosine triphosphate (GTP). When chloride was replaced by organic anions such as acetate, aspartate, or gluconate the speed of hyperpolarization diminished and the dark voltage of the rods was stabilized. The extent of stabilization correlates with the molecular weight of the anions. A significant difference in the stabilizing effect was found for L-aspartate and D-aspartate, suggesting an additional influence of the chirality. Effects of some of the anions on the configuration of the light responses were also observed.
Animals↗
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.
Animals↗
Enzyme treatment of photoreceptors: effects on the scotopic PIII component of the frog electroretinogram.
Enzymes are often used for preparation of excitable tissues. The effects of papain, trypsin, pronase, collagenase and hyaluronidase on the photoreceptor function were studied by recording of scotopic PIII responses. Each enzyme treatment diminished the amplitudes of the PIII responses with a characteristic time course. The effects of papain, collagenase and hyaluronidase were at least partly reversible, while trypsin and pronase irreversibly reduced the amplitudes of the PIII responses.
Animals↗