Circadian phase-dependent modulation of cGMP-gated channels of cone photoreceptors by dopamine and D2 agonist

Abstract

The affinity of cGMP-gated ion channels (CNGCs) for cGMP in chick retinal cone photoreceptors is under circadian control. Here we report that dopamine (DA) and D2 receptor agonists evoke phase-dependent shifts in the affinity of CNGCs for activating ligand. Inside-out patch recordings from cultured chick cones were performed at circadian time (CT) 4-7 and CT 16-19 on the second day of constant darkness. Exposing intact cells to DA or the D2 agonist quinpirole for 2 hr before patch excision caused a significant increase in the K(D) for cGMP during the night (CT 16-19) but had no effect during the day (CT 4-7). DA or quinpirole treatment had no effect on the Hill slope or the average number of channels per patch. The effect of DA was blocked by the D2 antagonist eticlopride and was not mimicked by D1 agonists or blocked by D1 antagonists. By contrast, a brief (15 min) exposure to DA or quinpirole caused a decrease in K(D) during the subjective day and had no effect during the subjective night. Thus, the effect of D2 agonists depends on both the duration of agonist exposure and the time of day. Application of DA or quinpirole evoked a transient activation of the MAP kinase Erk (extracellular signal-related kinase) during the day but caused a sustained inhibition during the night. Conversely, D2 agonists caused activation of Ca2+/calmodulin-dependent protein kinase II during the night and inhibited this enzyme during the day. A circadian oscillator in cones appears to regulate the nature of the transduction cascade used by D2 receptors.

Fig. 1.

Circadian rhythm in the apparent affinity of cGMP-gated channels for activating ligand in chick retinal cone photoreceptors. Cultured chick cones were entrained to 12 hr LD cycles for 4–5 d in vitro and then switched to DD. On the second day of DD, inside-out patches were excised from cones during the subjective day (CT 4–7) or the subjective night (CT 16–19). cGMP concentration–response curves were generated immediately after patch excision. A, Typical cGMP concentration–response curves obtained from patches excised during the subjective day (■) and subjective night (▪). Curves are shown with superimposed least-squares fits to the Hill equation. B, MeanK_D values from patches excised at CT 4–7 (n = 8 patches) and CT 16–19 (n = 9 patches) in cells free-running on the second day of DD. In this and all subsequent figures, error bars represent SEM, numbers in parentheses are the number of patches tested, and asterisks indicate p < 0.05.

Fig. 2.

Modulation of cGMP-gated channels by DA is mediated by D2 receptors. Experiments were performed on LD entrained cones free-running on the second day of DD. All drugs were applied starting 1.5–2 hr before inside-out patch recordings were made at CT 4–7 and CT 16–19. Agonists and antagonists were dissolved in a vehicle containing 0.4% ascorbic acid. A, Dopamine significantly increased the K_D of cGMP-gated channels during the subjective night (CT 16–19) but not the subjective day (CT 4–7) compared with the controls. A similar phase-dependent effect was produced by the selective D2 agonist quinpirole (B). The D2 antagonist eticlopride (50 μm) blocked the effect of dopamine (C), but treatment with eticlopride by itself had no effect on cGMP-gated channels (D). The selective D1 antagonist SCH 23390 (50 μm) did not reverse the effect of dopamine (E), and the selective D1 agonist SKF 38393 (1 μm) had no effect on cGMP-gated channels (F). In this and subsequent figures, all data points are means of 8–10 patches and derived from at least three different cell preparations.

Fig. 3.

A selective D2 agonist modulates Erk and CaMKII activation in chick cones. Chick retinal cells free-running on the second day of DD were treated with the D2 agonist quinpirole (500 nm) or vehicle for 2 hr starting at CT 3 or CT 15. At CT 5 or CT 17, cells were lysed and harvested for immunoblot analysis of Erk and CaMKII phosphorylation using antibodies selective for the phosphorylated forms of these proteins, as well as with antibodies that recognize total Erk regardless of its phosphorylation state.A, Erk diphosphorylation is substantially greater during the subjective night (CT 17) than during the subjective day (CT 5) in control cells. Cells treated for 2 hr with the D2 agonist quinpirole exhibit diminished Erk diphosphorylation during both the subjective night (CT 17) and the subjective day (CT 5). B, CaMKII phosphorylation is greater during the subjective day (CT 5) than during the subjective night (CT 17) in control cells. Treatment with 500 nm quinpirole for 2 hr dampened the rhythm in CaMKII phosphorylation, which was decreased during both the subjective day (CT 5) and the subjective night (CT 17). In this figure, representative blots are shown above results of densitometric analysis of five repetitions of each experiment. Ordinates of the graphs represent signal for phosphorylated enzyme divided by total Erk.

Fig. 4.

The effects of DA on Erk and CaMKII phosphorylation are blocked by the D2 antagonist eticlopride. Vehicle, DA (500 μm), eticlopride (50 μm), or a combination of the two drugs was applied at CT 3 and CT 15, and cells were lysed and harvested at CT 5 and CT 17. Analyses of protein phosphorylation were performed by immunoblot analysis as described for Figure 3. Bar graphs represent results of five repetitions of each experiment, showing the ratio of phosphorylated enzyme divided by total Erk as determined by densitometry.

Fig. 5.

The effects of dopamine agonists depend on the duration of receptor activation. Cultured chick cones free-running on the second day of DD were treated with dopamine (500 nm), quinpirole (500 nm), or vehicle for 15 min or 2 hr before excision of inside-out patches and determination of channelK_D. A, Treatment with DA for 15 min caused a significant decrease in cGMP-gated channelK_D during the subjective day (CT 4–7) but had no effect during the subjective night (CT 16–19) compared with controls. In contrast, treatment with dopamine for 2 hr significantly increased the K_D of cGMP-gated channels during the subjective night (CT 16–19) but had no effect during the subjective day (CT 4–7) compared with the controls. B, A similar pattern is observed in cells treated with the D2 agonist quinpirole. All data points are means of 8–10 patches and are derived from at least three different cell preparations.

Fig. 6.

The time course of D2 receptor effects on Erk and CaMKII phosphorylation in chick photoreceptors. Cultured cone photoreceptors cells were treated with quinpirole (500 nm) for 0, 15, 30, 60, or 120 min starting at CT 5 or CT 17. Cells were then lysed and harvested immediately for immunoblot analysis of Erk and CaMKII phosphorylation. A, During the subjective day (CT 5), quinpirole treatment evoked a robust but transient increase in Erk phosphorylation, which peaked at 15 min and fell to slightly below the baseline after 2 hr of continuous treatment. In contrast, during the subjective night (CT 17), quinpirole treatment caused only the slowly developing but sustained decrease in Erk phosphorylation. Top panels show a typical immunoblot, and bottom panels are densitometric analyses of several repetitions of these experiments. B, Treatment with quinpirole evoked a transient increase of CaMKII phosphorylation during the subjective night (CT 17), which peaked at 15 min and gradually fell to baseline after 2 hr. In contrast, during the subjective day (CT 5), quinpirole treatment evoked only the slowly developing inhibition of CaMKII phosphorylation.

Fig. 7.

Exposure to DA for 2 hr does not affectcPer2 mRNA levels in embryonic chick retina.A, Expression of cPer2 mRNA is rhythmic in chick retina. Whole retinas from entrained chick embryos were homogenized, and cPer2 transcript levels were quantified by Q-PCR. cPer2 is high during the subjective day and low during the subjective night. B, Cultured cone photoreceptors were treated with 500 nm dopamine or vehicle for 2 hr starting at CT 3 or CT 15 on the second day of DD after 5 d of LD, and cPer2 mRNA was measured by Q-PCR at CT 5 and CT 17. Dopamine does not affect the circadian oscillation of cPer2 in cultured chick photoreceptors.

Fig. 8.

The effects of dopamine on cGMP-gated channels are not mediated by inhibition of cAMP production and are not sensitive to pertussis toxin. A, Control cells exhibit a robust circadian modulation of channel K_D. Treatment with 500 μm 8-CPT-cAMP (cAMP), a membrane-permeable analog of cAMP, caused a significant decrease inK_D during the subjective day (CT 4–7) but not during the subjective night (CT 16–19) compared with controls. DA (500 nm) treatment evoked the opposite effect; i.e., there was a significant increase in K_D during the subjective night but no effect during the subjective day. Treatment with DA (500 nm) in the presence of 500 μm8-CPT-cAMP (Dopamine + cAMP) resulted in a significant increase inK_D during the subjective night but no apparent effect during the subjective day; i.e., the effects were indistinguishable from those of DA alone. In this experiment, drugs were applied 1.5–2 hr before inside-out patch recordings from cultured cones on the second day of DD. B, After pretreatment with 200 ng/ml pertussis toxin (PTX) for 18–24 hr, a subsequent 2 hr exposure to DA evoked a modulation of cGMP-gated channels comparable with that observed in control cells. Pertussis toxin by itself had no effect on the affinity of cGMP channels. All data points are means of 8–10 patches and are derived from at least three different cell preparations.