Tonic dopamine inhibition of L-type Ca2+ channel activity reduces alpha1D Ca2+ channel gene expression

Abstract

Hormones and neurotransmitters have both short-term and long-term modulatory effects on the activity of voltage-gated Ca2+ channels. Although much is known about the signal transduction underlying short-term modulation, there is far less information on mechanisms that produce long-term effects. Here, the molecular basis of long-lasting suppression of Ca2+ channel current in pituitary melanotropes by chronic dopamine exposure is examined. Experiments involving in vivo and in vitro treatments with the dopaminergic drugs haloperidol, bromocriptine, and quinpirole show that D2 receptors persistently decrease alpha1D L-type Ca2+ channel mRNA and L-type Ca2+ channel current without altering channel gating properties. In contrast, another L-channel (alpha1C) mRNA and P/Q-channel (alpha1A) mRNA are unaffected. The downregulation of alpha1D mRNA does not require decreases in cAMP levels or P/Q-channel activity. However, it is mimicked and occluded by inhibition of L-type channels. Thus, interruption of the positive feedback between L-type Ca2+ channel activity and alpha1D gene expression can account for the long-lasting regulation of L-current produced by chronic activation of D2 dopamine receptors.

Fig. 1.

Haloperidol treatment in vivo elevates NIL α_1D mRNA but not α_1C or α_1A mRNAs. A, Representative autoradiographs of RNase protection assays designed to measure α_1D and α_1C (top) or α_1A (bottom) mRNAs, along with β-actin or cyclophilin mRNA for normalization. Gel lanes contain the following:Y, yeast RNA (50 μg); B,C, and H, total NIL RNA from bromocriptine, vehicle, and haloperidol treatment groups, respectively.B, Effect of 6 hr in vivo dopaminergic drug treatment on α_1D (top;n = 6), α_1C (middle;n = 3), and α_1A(bottom; n = 4) mRNA levels. Quantitation was performed by phosphorimager analysis using short (1–3 hr) gel exposures. Note that only the effect of haloperidol on α_1D mRNA was statistically significant.

Fig. 2.

Chronic quinpirole treatment in vitro downregulates melanotrope α_1D mRNA but not α_1C mRNA. A, Representative autoradiograph of an RNase protection assay measuring α_1D and α_1C mRNAs. Gel lanes contain the following:Y, yeast RNA (50 μg); C andQ, total RNA from melanotropes cultured in control media or media with quinpirole (1 μm), respectively.B, Effect of 4 d quinpirole treatment on α_1D (n = 15) and α_1C(n = 14) mRNA levels.

Fig. 3.

Chronic quinpirole treatment in vitro induces a long-lasting suppression of total Ca²⁺ channel currents in melanotropes.A, Currents recorded in the presence of 1 μm Bay K 8644 using 100 msec depolarizations from a holding potential of −50 mV to several potentials from a representative control cell. B, Plot of peak currents (normalized to membrane capacitance) recorded during 100 msec depolarizations to various potentials versus voltage in melanotropes cultured in the absence or presence of quinpirole for 5–10 d. Data inA and B were obtained from melanotropes dissociated by trypsin–viokase digestion (see Materials and Methods).

Fig. 4.

Chronic quinpirole treatment in vitro induces a long-lasting suppression of L-type Ca²⁺ channel current density without changing its functional properties. A, Illustration of the method of isolating L-channel tail current from total Ca²⁺channel current in melanotropes (see Results for details). Noisy traces are 10 msec portions of tail currents recorded at −50 mV after a step depolarization to +75 mV. The smooth traces are exponential curves fit to the currents. The time constants are 0.16 msec (monoexponential curve in top) and 2.5 and 21.9 msec (biexponential curve in bottom). Thearrows are placed at 2.4 msec after repolarization to −50 mV. B, Chronic quinpirole treatment does not alter L-channel deactivation properties. Data were obtained with biexponential curve-fitting analysis of Bay K 8644-slowed tail currents in control and quinpirole-treated cells. Bars on theleft and right halves of the graph correspond to the left and right y-axes, respectively.C, Chronic quinpirole treatment does not alter the voltage-dependence of activation of L-channels. Normalized conductance (G) versus step depolarization potential (Vm) data for control and quinpirole-treated cells are fitted with Boltzmann equations (smooth curves). D, Maximal L-current density in melanotropes cultured for 6–10 d in control or quinpirole-containing media. Maximal L-current density values were 46.3 ± 6.6 pA/pF in control cells and 30.1 ± 4.1 pA/pF in quinpirole-treated cells. Data in B–D come from 9 control cells and 13 quinpirole-treated cells.

Fig. 5.

Br-cAMP fails to prevent dopaminergic downregulation of α_1D mRNA. Effect of 4 d treatment with 1 μm quinpirole on α_1D mRNA levels in melanotropes cultured in the absence (control) or presence of 1 mm Br-cAMP (n = 3). Note that Br-cAMP had no statistically significant effect on quinpirole-induced downregulation of α_1D mRNA (downregulation in the absence and presence of Br-cAMP was 34 ± 5 and 47 ± 4%, respectively; p > 0.05 for the comparison between these two percentage downregulation values).

Fig. 6.

Inhibition of L-channels, but not P/Q-channels, mimics and occludes D2 receptor-induced downregulation of α_1D mRNA. α_1D mRNA levels in control and quinpirole-treated melanotropes cultured for 4 d in control media (No blockers; n = 15) or in the absence or presence of the indicated Ca²⁺ channel blockers (n = 3 in all cases). ns, Not statistically significant. The dashed line indicates the level of α_1D mRNA in quinpirole-treated cells cultured in the absence of Ca²⁺ channel blockers. Note that the L-channel inhibitor nimodipine mimics and occludes the effect of the D2-receptor agonist quinpirole. In contrast, P/Q-channel blockers have no effect.

Fig. 7.

Model of long-lasting suppression of L-current induced by D2 receptor-triggered interruption of positive feedback between L-channel activity and α_1D gene expression. See Discussion for detailed explanation. Dopamine occupation of D2 receptors induces hyperpolarization and inhibition of spontaneous action potential activity (both indicated by line drawings labeled membrane potential). Haloperidol antagonizes activation of D2 receptors by endogenous dopamine, thus allowing spontaneous action potential firing. L- and P/Q-channels are activated by the depolarization of action potentials. L-channel activity triggers both secretion and α_1D gene expression. P/Q-channels may also trigger secretion in melanotropes.