Antidepressant stimulation of CDP-diacylglycerol synthesis does not require monoamine reuptake inhibition

Abstract

Background: Recent studies demonstrate that diverse antidepressant agents increase the cellular production of the nucleolipid CDP-diacylglycerol and its synthetic derivative, phosphatidylinositol, in depression-relevant brain regions. Pharmacological blockade of downstream phosphatidylinositide signaling disrupted the behavioral antidepressant effects in rats. However, the nucleolipid responses were resistant to inhibition by serotonin receptor antagonists, even though antidepressant-facilitated inositol phosphate accumulation was blocked. Could the neurochemical effects be additional to the known effects of the drugs on monoamine transmitter transporters? To examine this question, we tested selected agents in serotonin-depleted brain tissues, in PC12 cells devoid of serotonin transporters, and on the enzymatic activity of brain CDP-diacylglycerol synthase - the enzyme that catalyzes the physiological synthesis of CDP-diacylglycerol.

Results: Imipramine, paroxetine, and maprotiline concentration-dependently increased the levels of CDP-diacylglycerol and phosphatidylinositides in PC12 cells. Rat forebrain tissues depleted of serotonin by pretreatment with p-chlorophenylalanine showed responses to imipramine or maprotiline that were comparable to respective responses from saline-injected controls. With fluoxetine, nucleolipid responses in the serotonin-depleted cortex or hippocampus were significantly reduced, but not abolished. Each drug significantly increased the enzymatic activity of CDP-diacylglycerol synthase following incubations with cortical or hippocampal brain tissues.

Conclusion: Antidepressants probably induce the activity of CDP-diacylglycerol synthase leading to increased production of CDP-diacylglycerol and facilitation of downstream phosphatidylinositol synthesis. Phosphatidylinositol-dependent signaling cascades exert diverse salutary effects in neural cells, including facilitation of BDNF signaling and neurogenesis. Hence, the present findings should strengthen the notion that modulation of brain phosphatidylinositide signaling probably contributes to the molecular mechanism of diverse antidepressant medications.

Figure 1

Effects of PCPA-induced serotonin depletion on antidepressant stimulation of CDP-diacylglycerol synthesis in brain tissue. Brain slices were prepared from the frontal cortex or hippocampus tissues of saline-injected control rats or animals that had been administered p-chlorophenylalanine (PCPA) to deplete endogenous serotonin stores. The tissue slices were labeled with [³H]cytidine and then incubated with predetermined concentrations of the antidepressant drugs fluoxetine (FLX, 100 μM), imipramine (IMI, 300 μM), or maprotiline (MAP, 100 μM). After 90 min, accumulated [³H]CDP-diacylglycerol was measured. Data were calculated as percentages relative to the respective basal accumulations in the control or PCPA group. Each bar represents the mean ± SEM (N = 6). PCPA treatment had no significant effect on basal [³H]CDP-diacylglycerol accumulation in either tissue; such basal effects being, respectively, 8194+806 v. 8555+1657 for control versus PCPA in frontal cortex, and 8026+1295 v. 8139+1642 for control versus PCPA in hippocampus. Each drug induced significant accumulations of [³H]CDP-diacylglycerol in either the control or PCPA tissues (ANOVA, p < 0.0001 for each drug). PCPA partially reduced FLX and MAP effects but did not eliminate any of the drug effects: ***p < 0.001, compared by posthoc Tukey tests.

Figure 2

Effects of antidepressant agents on [³H]CDP-diacylglycerol synthesis in PC12 cells. PC12 cells were prelabeled with [³H]cytidine for 30 min and cells in different culture wells incubated with various concentrations of the antidepressant drugs imipramine, paroxetine or maprotiline. [³H]CDP-diacylglycerol was determined after 3 h. Data were calculated as percentages relative to the control incubations that received no drug treatment. Each bar represents the mean ± SEM (N = 3 experiments). The drug treatments increased [³H]CDP-diacylglycerol significantly (ANOVA, p < 0.0001 for each drug). Posthoc Dunnett tests showed statistically significant increases in CDP-diacylglycerol at 30 and 100 μM. *p < 0.05; ***p < 0.001, compared to controls.

Figure 3

Effects of antidepressant agents on phosphatidylinositide synthesis in PC12 cells. PC12 cells were labeled with [³H]inositol and cells in different culture wells incubated with various concentrations of selected antidepressant drugs. After 3 h, accumulated [³H]phosphatidylinositides were determined and the data calculated as percentages relative to control. Each bar represents the mean ± SEM (N = 3 experiments). Each drug treatment increased the levels of [³H]phosphatidylinositides significantly and concentration-dependently (ANOVA, P < 0.0001 for each drug). Posthoc Dunnett tests showed statistically significant increases in phosphatidylinositides at 1-30 μM for imipramine and at 3-30 μM for maprotiline. *p < 0.05; ** P < 0.01; ***p < 0.001; compared to controls.

Figure 4

Antidepressant drug effects on CDP-diacylglycerol synthase activity in mouse cortex tissues. The enzymatic activity of CDP-diacylglycerol synthase (CDS) was measured in mouse cortical tissue preparations by determining the incorporation of radiolabeled cytidine derived from 5- [³H]CTP into CDP-diacylglycerol. Antidepressant agents were tested at concentrations ranging from 1 μM to 100 μM. Data from each assay were normalized against the control samples by calculating the percentages shown. Each bar is the mean ± SEM (N = 3 experiments). One-Way ANOVA analyses for each drug showed significant effects for imipramine (p < 0.001), maprotiline (p < 0.0001), and paroxetine (p < 0.01). Posthoc Dunnett tests showed that imipramine and maprotiline elicited significant effects at 3-100 μM concentrations while with paroxetine significant effects were evident at concentrations of 3-30 μM. *p < 0.05; ** P < 0.01; ***p < 0.001, compared to controls.