Cocaine- and amphetamine-regulated transcript peptide modulation of voltage-gated Ca2+ signaling in hippocampal neurons

Abstract

Administration of cocaine and amphetamine increases cocaine- and amphetamine-regulated transcript (CART) expression in the rat striatum (Douglass et al., 1995). CART mRNA is highly expressed in different parts of the human and rat brain, including hippocampus (Douglass et al., 1995; Couceyro et al., 1997; Kuhar and Yoho, 1999; Hurd and Fagergren, 2000). The presence of CART peptide 55-102 immunoreactivity in dense core vesicles of axon terminals suggests that the peptide may be released and may act as a neuromodulator (Smith et al., 1997) to induce neurophysiological and behavioral effects. Little is known, however, about CART peptide-responsive cells, receptor(s), or intracellular signaling mechanisms that mediate CART peptide action. Here we show that CART peptide 55-102 inhibits voltage-dependent intracellular Ca(2+) signaling and attenuates cocaine enhancement of depolarization-induced Ca(2+) influx in rat hippocampal neurons. The inhibitory effect of CART peptide 55-102 on Ca(2+) signaling is likely mediated by an inhibition of L-type voltage-gated Ca(2+) channel activity via a G-protein-dependent pathway. These results indicate that voltage-gated Ca(2+) channels in hippocampal neurons are targets for CART peptide 55-102 and suggest that CART peptides may be important in physiology and behavior mediated by the hippocampus, such as certain forms of learning and memory.

Fig. 1.

CART peptide 55–102 reduces the depolarization-induced Ca²⁺ signal.A, Cytosolic Ca²⁺ signal recorded from fura-2-loaded neurons before, during, and after bath application of CART peptide 55–102 (1 μm). High K⁺ (35 mm) medium application is indicated by the short bars, and CART peptide 55–102 application is indicated by the long bar.B, Dose–response relationship between CART peptide 55–102 concentration and its inhibitory effect on Ca²⁺ signal amplitude (n = 4).C, The effect of CART peptide 55–102 was abolished by previous incubation with DTT. D, Ca²⁺signals in undifferentiated PC12 cells before, during, and after CART peptide 55–102 application.

Fig. 2.

CART peptide 55–102 modifies Ca²⁺ influx in hippocampal neurons.A, Depolarization-induced Ca²⁺signals in hippocampal neurons in the absence and presence of 1 μm CART peptide 55–102 are shown superimposed.B, Effects of CART peptide 55–102 (1 μm) on depolarization-induced Ca²⁺ signals in the presence of 1 μm TG. C, Effects of CART peptide 55–102 on depolarization-induced Ca²⁺signals in the presence of 50 μm nifedipine. Nifedipine markedly reduced the Ca²⁺ signal, and the subsequent application of CART peptide 55–102 did not further inhibit the Ca²⁺ signals. The cells were also pretreated with TG. D, Depolarization-induced Ca²⁺signals in the presence of 5 μm Bay K 8644 and CART peptide 55–102 in TG-pretreated cells.

Fig. 3.

CART peptide 55–102 reduces L-type channel activity. A, Representative openings elicited by pulses from −50 to −5 mV in the control condition (left) and in the presence of the CART peptide (1 μm;right). The corresponding ensemble averages are shown at the bottom. B, Relative open probability (n · P_o) during a representative experiment. The openings were elicited by pulses to −5 mV every 6 sec. The n · P_ovalue was calculated for each depolarization epoch, and the values are plotted as a function of the epoch number. The CART peptide application period is indicated by the gray horizontal bar. C, Representative openings elicited by pulses from −50 to −5 mV in the presence of Bay K 8644 (2 μm; left) as control and after application of CART peptide 55–102 (1 μm; right).D, Ratios of the relative open probability (n · P_o) after application of the CART peptide (1 μm) to the respective control relative open probability are displayed using a box plot (left). The mean and median values in the control condition were 0.14 and 0.11, respectively. The shaded area represents the 95% confidence interval of the median. The ratios of the mean open duration (Dur) before and after the CART peptide application are also shown using a box plot (right). The mean and median values in the control condition were 0.72 and 0.72 msec, respectively. E, In the presence of Bay K 8644 (2 μm), the ratios of the open probability (n · P_o) before and after the CART peptide application are displayed using a box plot (left). The mean and median values in the presence of Bay K 8644 before CART application were 0.44 and 0.54, respectively.Right, Ratios of the mean open duration before and after the CART peptide application. The mean and median values in the presence of Bay K 8644 before CART application were 6.8 and 7.7 msec, respectively.

Fig. 4.

Signal transduction pathways mediating CART peptide 55–102 action. A, Ca²⁺signals in response to high-K⁺-induced depolarization were measured using fura-2 fluorescence in TG-pretreated rat hippocampal cells. Ca²⁺ signals are shown in the presence of 100 nm staurosporine, after a 5 min pretreatment, and then after addition of 1 μm CART peptide. B, Ca²⁺ transients in neurons pretreated with 5 μm genistein with and without CART peptide 55–102. C, Ca²⁺ signals after a 5 min pretreatment and in the continuous presence of 5 μm cyclosporine A and after addition of CART peptide.D, Ca²⁺ transients in hippocampal neurons pretreated with pertussis toxin (PTX; 50 ng/ml, 18 hr) and in the presence of CART peptide.

Fig. 5.

CART peptide decreases sensitivity of hippocampal neurons to cocaine. A, K⁺depolarization-induced Ca²⁺ transients in the presence of 0 and 1 μm cocaine (TG-pretreated cells).B, Increase in total Ca²⁺ influx in response to K⁺ depolarization in the presence of increasing cocaine concentrations in control conditions (open symbols) and after a single 5 min application of 1 μm CART peptide (filled symbols) in TG-pretreated hippocampal cells. C, The depolarization-induced Ca²⁺ transient is reduced in the presence of 1 μm CART peptide 55–102 and partially restored by 5 μm cocaine.