The effects of dopamine and dopamine receptor agonists on the phototransduction cascade of frog rods

Abstract

Purpose: Accumulating evidence suggests that dopamine, the major catecholamine in the vertebrate retina, may modulate cAMP-mediated signaling in photoreceptors to optimize vision in the light/dark cycle. The main putative mechanism of dopamine-induced adaptation changes in photoreceptors is activation of D₂-like receptors (D₂R), which leads to a decrease of the intracellular cAMP level and reduction of protein kinase A (PKA) activity. However, the mechanisms by which dopamine exerts its regulating effect on the phototransduction cascade remain largely unknown. The aim of the present study was to investigate the effects of dopamine and dopamine receptor agonists on rod photoresponses.

Methods: The experiments were performed on solitary rods of the Rana ridibunda frog. Photoreceptor currents were recorded using a suction pipette technique. The effects of dopamine (0.1-50 µM) and selective dopamine receptor agonists-D₁R agonist SKF-38393 (0.1-50 µM), D₂R agonist quinpirole (2.5-50 µM), and D₁-D₂ receptor heterodimer agonist SKF-83959 (50 µM)-were examined.

Results: We found that, when applied to the rod inner segments (RISs), dopamine and dopamine receptor agonists had no effect on photoresponses. In contrast, the rods responded to dopamine and all agonists applied to their outer segments by decreasing sensitivity to light. At the highest tested concentration (50 µM), the most prominent effect on light sensitivity was induced by D₁R agonist SKF-38393, while dopamine, D₂R agonist quinpirole, and D₁-D₂ receptor heterodimer agonist SKF-83959 produced somewhat lower and approximately equal effects. Moreover, SKF-38393 reduced sensitivity at all tested concentrations starting from the smallest one (0.1 µM), whereas dopamine and quinpirole started their action from the higher concentrations of 2.5 µM and 50 µM, respectively. In addition, dopamine, SKF-38393, and quinpirole, on average, did not change the intracellular calcium level as judged from the "exchange current", while SKF-83959 increased it by ~1.3 times.

Conclusions: Dopamine induces a decrease in rod sensitivity, mostly by reducing the activation rate of the cascade, and to a much lesser extent, speeding up the turning off of the cascade. The sign of the reaction to all tested drugs, lack of selectivity of dopamine and dopamine receptor agonist action, and analysis of factors that determine sensitivity of photoreceptors suggest that, in rod outer segments (ROSs), dopamine action is mediated by D₁-D₂ receptor heterodimers but not D₁R or D₂R alone. This work supports the assumption made earlier by other authors that dopamine exercises its regulatory effect via at least two independent mechanisms, which are cAMP and Ca²⁺ mediated.

Figure 1

Effect of dopamine on the light sensitivity of frog rods in the “outer segment out” configuration. A: Fractional responses of a frog rod in Ringer’s solution (blue solid line, labeled “R”) just before the start of dopamine application and after 20 min of exposure to 50-µM dopamine-containing solution (red solid line, labeled “D”). Each trace is an average of 10 responses. The responses were normalized to a magnitude of the corresponding dark current that was 22.5 pA in Ringer’s solution and 21 pA at the end of perfusion with dopamine. The flash intensity in these records was 0.81 photon ・μm⁻² per flash. B: Changes of the sensitivity presented as a ratio of fractional photoresponses after 20 min of incubation in dopamine-containing solution to fractional photoresponses in Ringer’s solution. Control data are depicted as a ratio of fractional photoresponses after turning on a jet of drug-free Ringer’s solution to fractional photoresponses before introducing rods into the jet. Average from 7–12 cells in each group. Error bars represent the standard error of the mean (SEM); * denotes p<0.05 versus control. The significance of differences between different concentrations of dopamine is indicated by connecting lines between bars; # denotes p<0.05 between concentrations of dopamine.

Figure 2

Procedure for determining kinetics of the rising and falling phases of rod photoresponses to weak light flashes (the same cell as in Figure 1A). A: The rising phase of fractional responses recorded in Ringer’s solution (blue solid line, labeled “R”) and in the presence of 50-µM dopamine (red solid line, labeled “D”) after 20 min of exposure. The red dashed line shows the response in dopamine-containing solution scaled up by a factor of 1.27 to coincide with the response in Ringer’s solution. B: A single-exponential fit of the falling phase of fractional responses in Ringer’ solution (blue solid line, labeled “R”) and after 20 min incubation in 50-µM dopamine-containing solution (red solid line, labeled “D”). The turn-off time constants (τ_off) are shown near the curves of photoresponses.

Figure 3

Determining the dominant time constant of recovery of rods from saturating flashes. A: The set of responses to saturating stimuli of increasing flash intensities was recorded in Ringer’s solution. The same set of responses was recorded after 20 min from the start of dopamine perfusion (data not shown). The flash intensities in these records were 153–3,431 photons ・μm⁻² per flash. B: Plotting the time of recovery from saturation (T_rec) as a function of the flash intensity in log scale in Ringer’s solution (■) and after 20 min of incubation in 2.5-µM dopamine-containing solution (□). The straight lines approximate experimental points with T_rec = C + τDˑln(I). T_rec is defined as the time of regaining 20% of the dark current (see Figure 3A). The dominant recovery time constants (τ_D) are shown near the lines.

Figure 4

Analysis of kinetics of light-induced calcium changes in rod outer segment in Ringer’s solution. A: An averaged response of four individual saturated responses. Each response was normalized to the corresponding dark current (~23 pA) and the average plotted “upside down” to show the circulating current. The flash intensities in these recordings were 2,220–3,520 photons ・ μm⁻² per flash. B: The same response as in Figure 4A on an enlarged scale. The noisy black line shows the averaged response. The smooth red line is a single-exponential approximation.

Figure 5

Effects of the D₁R agonist SKF-38393 on the basic parameters of rod photoresponses in “outer segment out” configuration. Changes of the parameters presented as a ratio of responses after 20 min of exposure to SKF-38393 to photoresponses in Ringer’s solution. Control (0 µM) data are depicted as a ratio of photoresponses after turning on a jet of drug-free Ringer’s solution to photoresponses before introducing rods into the jet. Effects of SKF-38393 on the light sensitivity (A), activation rate (B), turn-off time constant (C), dark current (D), and dominant recovery time constant (E). Average of 8–10 cells in each group. Error bars represent the standard error of the mean (SEM); * denotes p<0.05 versus control. The significance of differences between different concentrations of the D₁R agonist is indicated by connecting lines between bars; # denotes p<0.05 between concentrations of the D₁R agonist.

Figure 6

Correlation of changes of light sensitivity and the activation rate in individual cells. On the Х-axis, the relative sensitivity of a cell to light is plotted; the Y-axis shows the relative activation rate of a rod. Each point represents one cell. A: Correlation graph for dopamine (at 0.1–50 µM; n = 37). B: Correlation graph for the D₁R agonist SKF-38393 (at 0.1–50 µM; n = 38). C: Correlation graph for D₂R agonist quinpirole (at 2.5–50 µM; n = 30). D: Correlation graph for D₁–D₂ receptor agonist SKF-83959 (at 50 µM; n = 10).

Figure 7

Correlation of changes of light sensitivity and the time of photoresponse turn off in individual cells. On the Х-axis, the relative sensitivity of a cell to light is plotted, while the Y-axis shows the relative turn-off time constant of a rod. Each point represents one cell. A: Correlation graph for dopamine (at 0.1–50 µM; n = 37). B: Correlation graph for D₁R agonist SKF-38393 (at 0.1–50 µM; n = 38). C: Correlation graph for D₂R agonist quinpirole (at 2.5–50 µM; n = 30). D: Correlation graph for D₁–D₂ receptor agonist SKF-83959 (at 50 µM; n = 10).

Figure 8

Correlation of changes of light sensitivity and the size of “exchange current” in individual cells. On the Х-axis, the relative sensitivity of a cell to light is plotted; the Y-axis shows the relative “exchange current” magnitude of a rod. Each point represents one cell. A: Correlation graph for dopamine (at 50 µM; n = 6). B: Correlation graph for D₁R agonist SKF-38393 (at 50 µM; n = 7). C: Correlation graph for D₂R agonist quinpirole (50 µM; n = 5). D: Correlation graph for D₁–D₂ receptor agonist SKF-83959 (at 50 µM; n = 5).

Figure 9

Correlation of changes of light sensitivity and the time constant of “exchange current” in individual cells. On the Х-axis, the relative sensitivity of a cell to light is plotted; the Y-axis shows the relative “exchange current” magnitude of a rod. Each point represents one cell. A: Correlation graph for dopamine (at 50 µM; n = 6). B: Correlation graph for D₁R agonist SKF-38393 (at 50 µM; n = 7). C: Correlation graph for D₂R agonist quinpirole (50 µM; n = 5). D: Correlation graph for D₁–D₂ receptor agonist SKF-83959 (at 50 µM; n = 5).