Differential regulation of the high affinity choline transporter and the cholinergic locus by cAMP signaling pathways

Abstract

Synthesis, storage and release of acetylcholine (ACh) require the expression of several specialized enzymes, including choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT) and the high-affinity choline transporter (CHT). Extracellular factors that regulate CHT expression and their signaling pathways remain poorly characterized. Using the NSC-19 cholinergic cell line, derived from embryonic spinal cord, we compared the effects of the second messenger cAMP on the expression of CHT and the cholinergic locus containing the ChAT and VAChT genes. Treatment of NSC-19 cells with dbcAMP and forskolin, thus increasing intracellular cAMP levels, significantly reduced CHT mRNA expression, while it upregulated ChAT/VAChT mRNA levels and ChAT activity. The cAMP-induced CHT downregulation was independent of PKA activity, as shown in treatments with the PKA inhibitor H-89. The alternative Epac-Rap pathway, when stimulated by a specific Epac activator, led to significant downregulation of CHT and ChAT, and, to a lesser extent, VAChT. In contrast, the PKA activator 6-BNZ-cAMP stimulated the expression of all three genes, but with varying concentration-dependence profiles. Our results indicate that elevations of intraneuronal cAMP concentration have differential effects on the cholinergic phenotype, depending on the involvement of different downstream effectors. Interestingly, although CHT is expressed predominantly in cholinergic cells, its regulation appears to be distinct from that of the cholinergic locus.

Figure 1. cAMP induces neurite outgrowth and produces differential regulation of CHT and the cholinergic locus expression in NSC-19 cells

(A) Phase-contrast microscope pictures were taken after 36 h of treatment. Objective lens 20 x. Size bars: 100 μM. (B) ChAT activity was assayed in cell extracts as described in Experimental Procedures. Data are shown in the bar graph as means ± SEM of two independent cultures (each experiment was performed in duplicate). (*) significantly different from corresponding untreated control. (C) Total RNA preparations were subjected to RT-PCR reactions with CHT, ChAT and VAChT primers. RT-PCR with β-actin primers served as a loading control. PCR product band intensities were quantified by the Kodak Image Station Software. Results are expressed as means ± SEM of three experiments. (*) significantly different from corresponding untreated control.

Figure 2. Effect of forskolin on CHT and VAChT mRNA expression in NSC-19 cells

(A) NSC-19 cells were treated with increasing concentrations of forskolin, as indicated, for 72 h. Total RNA preparations were subjected to RT-PCR reactions with CHT and β-actin primers. Band intensities were quantified by the Kodak Image Station Software. Each treatment was performed in duplicate, hence it is represented by two lanes. Data are shown in the linear graph as means ± range of two samples. (B) The cells were stimulated with 30 μM forskolin for 48 h. RT-PCR reactions were done on total RNA preparations with CHT, ChAT, VAChT and β-actin primers. Band intensities were quantified by the Kodak Image Station Software and presented as means ± SEM; n (CHT) = 12; n (ChAT) = 8; n (VAChT) = 10. (*) significantly different from corresponding untreated control (paired t-test).

Figure 3. Forskolin-induced upregulation of the cholinergic locus, but not the downregulation of CHT expression, depends on the PKA pathway

NSC-19 cells were pre-treated with 20 μM H-89 for 1 h, and then stimulated with 30 μM forskolin for 72 h. (A) Phase-contrast microscope pictures were taken after 24 h of treatment. Objective lens 20 x. Size bars: 100 μM. (B) RT-PCR reactions with CHT, VAChT, ChAT and β-actin primers were performed on total RNA preparations. Each treatment is represented by two lanes. Band intensities were determined by the Kodak Image Station Software. Data are expressed as means ± SEM of three independent experiments (each in duplicate). (*) statistically different from the corresponding untreated control.

Figure 4

Morphology of NSC-19 cells treated with various inducers of cAMP signaling. The cells were cultured in the presence of 30 μM adenylyl cyclase activator forskolin, 100 μM PKA activator 6-BNZ-cAMP, or 400 μM Epac activator 8-pCPT-2′-O-Me-cAMP. Phase-contrast microscope pictures were taken after 72 h of treatment. Objective lens 20 x. Size bars: 100 μM.

Figure 5. Effect of PKA and Epac activators on the expression of CHT and the cholinergic locus mRNAs in NSC-19 cells

(A) The cells were exposed to a range of concentrations of 6-BNZ-cAMP, for 72 h. Total RNA preparations were subjected to RT-PCR reactions with the CHT, VAChT, ChAT and β-actin primers and analyzed by gel electrophoresis. Band intensities were determined by the Kodak Image Station Software. The graph combines data from three experiments and presents relative mRNA expression for each gene as a function of 6-BNZ-cAMP concentration (logarithmic scale). (B) The cells were exposed to various concentrations of the Epac activator 8-pCPT-2′-O-Me-cAMP, as indicated, for 72 h. Total RNA preparations were subjected to RT-PCR reactions with the CHT, VAChT, ChAT and β-actin primers. Each treatment is represented by two lanes. Band intensities were determined by the Kodak Image Station Software. Data in the graph are expressed as means ± range of two samples.