Dopamine D4 receptors regulate intracellular calcium concentration in cultured chicken cone photoreceptor cells: relationship to dopamine receptor-mediated inhibition of cAMP formation

Abstract

Dopamine is a retinal neuromodulator secreted from amacrine and interplexiform cells. Activation of dopamine D4 receptors on photoreceptor cells reduces a light-sensitive pool of cAMP. The aim of the present study was to evaluate the role of dopamine receptors and cAMP in the regulation of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in photoreceptor cells of chick retina. Retinal cells from 6 day-old chicken embryos were isolated and cultured for 5-7 days prior to experiments. Cone photoreceptors were the predominant cell type in these cultures. Dopamine and agonists of dopamine D4 receptors suppressed K(+)-stimulated uptake of (45)Ca(2+) and [Ca(2+)](i), measured with the Ca(2+)-sensitive fluorescent dye fura-2AM. The effects of the agonists were blocked by dopamine D2/D4 receptor antagonists or by pertussis toxin. 8Br-cAMP, a cell-permeable analog of cAMP, had no effect on inhibition of K(+)-stimulated (45)Ca(2+) influx or [Ca(2+)](i) by dopamine D2/D4 receptor agonists. Quinpirole inhibited the increase in cAMP level elicited by K(+), which requires Ca(2+) influx through voltage-gated Ca(2+) channels, but not that induced by the calcium ionophore A23187. Moreover, dopamine had no effect on either forskolin-stimulated or Ca(2+)/calmodulin-stimulated adenylyl cyclase activity in cell membranes prepared from the cultured cells. These data indicate that the decrease of cAMP elicited by dopamine D4 receptor stimulation may be secondary to decreased [Ca(2+)](i).

Fig. 1

Dopamine and quinpirole inhibit calcium influx in chicken photoreceptor cell cultures. Cells were prepared and treated with drugs and ⁴⁵Ca²⁺ uptake measured during a 30 sec incubation as described in Experimental Procedures A. Dopamine inhibited calcium influx in cultured chicken photoreceptor cells. Elevated extracellular KCl (35 mM) significantly increased ⁴⁵Ca²⁺ influx in cultured chicken photoreceptor cells compared to basal conditions [3.6 mM KCl; **p<0.01]. Dopamine (1 μM) significantly reduced K⁺-stimulated ⁴⁵Ca²⁺ uptake [^††p<0.01 vs. vehicle (35 mM KCl)]. The effect of dopamine was blocked by 10 μM spiperone and 10 μM clozapine [**p<0.01 vs. 3.6 mM KCl]; n=6 per group. B. Quinpirole inhibits calcium influx in cultured chicken photoreceptor cells. KCl (35 mM) significantly increased ⁴⁵Ca²⁺ influx in cultured chicken photoreceptor cells compared to basal conditions [**p<0.01]. Quinpirole (0.3 μM) inhibited K⁺-induced ⁴⁵Ca²⁺ uptake [^††p<0.01 vs. vehicle]. The inhibitory effect of quinpirole was blocked by 10 μM spiperone, [**p<0.01 vs. vehicle] or pretreatment with 50 ng/ml pertussis toxin (PTX) [**p<0.01 vs. vehicle]; n=5–6 per group.

Fig. 2

Dopamine inhibits the depolarization-evoked increase in [Ca²⁺]_i in cultured chicken photoreceptor cells. A. Representative trace of [Ca²⁺]_i recording of identified photoreceptor cells treated with three repetitive stimulations with 35 mM KCl. K⁺-evoked depolarizations produce highly reproducible increases in [Ca²⁺]_i. B. Representative trace of [Ca²⁺]_i recording of identified photoreceptor cells treated with three repetitive stimulations with 35 mM KCl and dopamine (0.1 μM) added during the 2^nd stimulation (S2). C. Plots of S2/S1 ratios (S2 = peak area during the 2^nd stimulation, and S1 = peak area during the 1^st stimulation); n= 12 for controls; n=8 for dopamine; * p<0.05 vs control.

Fig. 3

Quinpirole inhibits depolarization-evoked increase in [Ca²⁺]_i in cultured chicken photoreceptor cells. A. Quinpirole (0.3 μM) significantly (**p<0.05 vs control, n=12) reduced the K⁺-evoked increase in [Ca²⁺]_i in chicken photoreceptor cells. Data expressed as S2/S1 ratio. Inhibitory effect of quinpirole was reduced by the D2/D4 Dopamine receptor antagonist 10 μM spiperone (p>0.05 vs control, n=4). SCH 23390 (10 μM, **p<0.05 vs control, n=16) failed to alter the inhibitory action of quinpirole.

Fig. 4

D4 receptor agonist, PD 168,077, inhibits depolarization-evoked increase in [Ca²⁺]_i in chicken photoreceptor cells. A. The inhibitory effect of PD 168,077 on [Ca²⁺]_i was concentration-dependent (0.025–1.0 μM), with significant inhibition at concentrations of 0.1 μM and above (** p<0.01). B. The inhibitory effect of 0.1 μM PD 168,077 (** p<0.01 vs. control; n=20) was blocked by 1.0 μM L 745,870 (^††p<0.01 vs. PD 168,077 n=9).

Fig. 5

Effect of 8Br-cAMP on depolarization-evoked ⁴⁵Ca²⁺ influx. KCl (35 mM) significantly increased ⁴⁵Ca²⁺ influx compared to basal conditions [** p<0.01 vs. 3.6 mM K⁺]; 0.3 μM quinpirole significantly reduced K⁺-stimulated ⁴⁵Ca²⁺ uptake [^†† p<0.01 vs. 35 mM KCl]. Pretreating with 2 mM 8Br-cAMP had no effect on K⁺-stimulated ⁴⁵Ca²⁺ influx in the absence (**p<0.01) or presence (^††p<0.01) of 0.3 μM quinpirole; n=4–5 per group.

Fig. 6

Lack of effect of 8Br-cAMP on the reduction of [Ca²⁺]_i elicited by PD168.077. Cells were pretreated for 1 hour with 3 mM 8Br-cAMP (Fig. 6B) or vehicle (Fig. 6A) prior to stimulation with 35 mM K⁺ for 13 min; 8Br-cAMP remained in the medium during the recordings. Seven minutes following the introduction of 35 mM K⁺, cells were treated with PD168,077 (0.5 μM) or vehicle (DMSO) and recordings were continued. To quantify the effect of drug treatment, baseline-subtracted [Ca²⁺]_i values were integrated for the 200 sec prior to addition (Area 1) and the 200 sec following addition (Area 2) of PD168,077 or vehicle. Area 2/Area 1 ratios were determined (Fig. 6C). PD168,077 significantly reduced [Ca²⁺]_i (p<0.01) and 8Br-cAMP had no significant effect on the response to the D4 receptor agonist. Samples sizes were 19 (DMSO), 24 (8Br-cAMP + DMSO), 16 (PD168,077), and 32 (8Br-cAMP + PD168,077). A positive control experiment was conducted to determine if 8Br-cAMP activated protein kinase A (PKA) under these conditions. Cells were analyzed for induction of arylalkylamine N-acetyltransferase (AANAT), a photoreceptor enzyme that is regulated by cAMP-dependent phosphorylation (Alonso-Gomez and Iuvone, 1995; Pozdeyev et al., 2006). Incubation with 8Br-cAMP significantly elevated AANAT activity (Control – 13 ± 0.6; 8Br-cAMP – 40 ± 1.5 pmol N-acetyltryptamine min⁻¹ mg protein⁻¹; p<0.01; N=5 per group).

Fig. 7

Lack of effect of quinpirole on adenylyl cyclase activity in membranes prepared from cultured chicken photoreceptor cells. Adenylyl cyclase activity (AC) in membranes was significantly increased by treatment with either 10 μM forskolin [n=3, **p<0.01 vs. Basal activity] or 120 nM Ca²⁺/calmodulin [n=3, **p<0.01 vs. basal activity]. 10 μM and 100 μM quinpirole failed to inhibit either forskolin-stimulated [n=3, **p<0.01 vs. basal activity] or Ca²⁺/calmodulin-stimulated adenylyl cyclase activity [n=3, **p<0.01 vs. basal activity].