D2-Like dopamine autoreceptor activation reduces quantal size in PC12 cells

Abstract

D2-like dopamine autoreceptors regulate dopamine release and are implicated in important actions of antipsychotic drugs and rewarding behaviors. To directly observe the effects of D2 autoreceptors on exocytic neurotransmitter release, we measured quantal release of dopamine from pheochromocytoma PC12 cells that express D2 and D4 autoreceptors. High potassium-evoked secretion in PC12 cells produced a unimodal population of quantal sizes. We found that exposures to the D2-like agonist quinpirole that inhibited tyrosine hydroxylase activity by approximately 50% also reduced quantal size by approximately 50%. The reduced quantal size was blocked by the D2 antagonist sulpiride and reversed by L-DOPA. Quinpirole also decreased the frequency of stimulation-evoked quantal release. Together, these findings indicate effects on quantal neurotransmission by D2-like dopamine autoreceptors previously distinguished as synthesis-modulating autoreceptors that regulate tyrosine hydroxylase activity versus impulse-regulating autoreceptors that modulate membrane potential. The results also provide an initial demonstration of a receptor-mediated mechanism that alters quantal size.

Fig. 1.

PCR analysis of D₂-like receptors in PC12 cells. RT-PCR analysis indicates that PC12 cells display message for D₂ (long and short forms) and D₄ DA receptors at the expected molecular weight (Materials and Methods). No D₃ message was observed. Ventral striatum (postnatal day 1) displays message for all three D₂-like receptors. The ladders on either side indicate 100 bp intervals for scaling. The 200 and 400 bp bands are indicated byasterisks.

Fig. 2.

Effect of quinpirole on TH activity. PC12 cultures were exposed to tyrosine (100 μm for 40 min) in the presence of the DOPA decarboxylase inhibitor brocresine (500 μm). After incubation and extraction, l-DOPA was measured by HPLC. Quinpirole decreased l-DOPA synthesis to 59% of controls. The inhibition was blocked by coincubation with the D₂-like antagonist sulpiride (40 μm).n = 5 for each condition; error bars indicate SEM; *p < 0.0001 by one-way ANOVA.

Fig. 3.

Effect of quinpirole on DA uptake. PC12 cultures were exposed to a combination of unlabeled and [³H]DA (300 fmol) in physiological medium for 5 min. DA uptake was halted by replacing with ice-cold physiological medium, and the cells were solubilized with PCA. Background DA uptake was determined in saline in which choline replaced sodium. Exposure to either sulpiride or quinpirole did not alter specific DA uptake.n = 5 for each condition; error bars indicate SEM.

Fig. 4.

Depolarization by high potassium elicits quantal release in PC12 cells. A, Amperometric spikes after PC12 stimulation with 80 mm KCl for 6 sec (applied atarrow). This representative control trace represents 15 spikes (I_max > 4.5 rms noise), ranging from 1.25 to 14.0 pA with a mean amplitude of 4.8 ± 1.0 pA and quantal size of 201,000 ± 39,400 molecules. B, The portion above the broken line in A is displayed with an expanded time resolution. The quantal size (#molecules), peak amplitudes (I_max; in picoamperes), and width at half height (t_½; in milliseconds) are displayed under four corresponding events. C, For the sample event indicated with the asterisk inB, the maximal amplitude is indicated by the top broken line, and the half height is indicated by themiddle broken line. The foot, filled by an array of points, is estimated by integrating the portion of the event preceding the slope (broken line) between the 60 and 90% rise-times (Chow and von Ruden, 1995). D, For control cells (events in Table 1), the interspike intervals (int_spike; in milliseconds) are displayed in 100 msec bins on the x-axis. The percentage of intervals within each bin (n/N) is labeled on the y-axis. The decreased representation at longer interval values is approximated by an exponential decay (see Materials and Methods).

Fig. 5.

Effects of quinpirole on quantal release. Examples of traces recorded from (A) a control cell and (B) a cell exposed to 0.5 μmquinpirole for 40 min. Cells were stimulated by 6 sec applications of 80 mm KCl at the arrow. Quinpirole decreased the mean quantal size of individual release events (Table 1).C, Distribution of mean quantal size values for all cells that displayed more than five events within 20 sec of stimulation (controls, n = 34; quinpirole,n = 18; sulpiride, n = 30; quinpirole/sulpiride, n = 13).D–G, The cumulative frequencies of interspike intervals are compared between treatments. Quinpirole shifted interspike values to longer durations. The effect was blocked by sulpiride (B).H–K, For each group, the distribution of quantal sizes (#molecules/1000) is indicated as a histogram of the untransformed quantal sizes (left column) and the log values of those quantal sizes (right column). Quinpirole shifted the frequency distribution of quantal sizes to the left. The lower limit of each bin size is displayed on the x-axis. The distribution of log transformations in each case is closely approximated by a normal distribution (r² ≥ 0.987; Table2).

Fig. 6.

Effect of l-DOPA on quinpirole action. Examples of traces recorded from a cell exposed to l-DOPA (50 μm for 40 min) (A) and a cell that has undergone identical l-DOPA exposure in the presence of quinpirole (0.5 μm for 40 min) (B). Cells were stimulated by 6 sec applications of 80 mm KCl at the arrow. C, Distribution of mean quantal size values for cells that displayed more than five events within 20 sec of stimulation (controls,n = 34; quinpirole, n = 18;l-DOPA, n = 25;l-DOPA–quinpirole, n = 19). The control and quinpirole-only groups are the same as those represented in Figure 5. l-DOPA increased the mean quantal size of both control and quinpirole-treated release events to a similar degree (Table 1). D–F, Cumulative frequencies of interspike intervals are compared between treatments.l-DOPA blocked the effect of quinpirole on interspike intervals. G–J, Distribution of quantal sizes indicated as histograms of log transforms of the quantal size as in Figure 5. For comparison, the control and quinpirole-treated groups are also shown. l-DOPA shifted the frequency distribution of quantal sizes to the right.