Relationship between dopamine agonist stimulation of inositol phosphate formation and cytidine diphosphate–diacylglycerol accumulation in brain slices

Dopamine receptor-coupled stimulation of inositol phosphate formation has been characterized extensively, but little is known about the diacylglycerol arm of this dual-signaling pathway. This study examined several parameters of cytidine diphosphate–diacylglycerol (CDP–DG) accumulation as an index of agonist-stimulated DG formation. Rat brain slices pre-labeled with 5-[³H]cytidine were incubated with various test agents in the presence of LiCl and accumulated CDP–DG analyzed. Dopamine and SKF38393 significantly and dose-dependently stimulated CDP–DG accumulation. SKF38393 responses were inhibited by neomycin and reversed by myo-inositol or by exclusion of LiCl. Compared to inositol phosphate formation in 2-[³H]inositol-prelabeled slices, the CDP–DG responses were proportionately greater, while the agonist EC₅₀ values were similar between the two assays. The D₁-receptor antagonist SCH23390 inhibited SKF38393-mediated responses at 0.1–10 μM concentrations, whereas greater concentrations reversed the inhibition. SKF38393 effects were completely blocked by the DG kinase inhibitor R59022, thus precluding any role for phospholipase-D or de novo phosphatidate synthesis in the dopaminergic response. D609 which inhibits phosphatidylcholine-specific phospholipase-C (PLC), potently inhibited both CDP–DG accumulation and inositol phosphate formation. These findings demonstrate that the selective D₁-receptor antagonist SCH23390 is a partial agonist at the D₁-like dopamine receptor that couples to phosphoinositide signaling, that dopaminergic facilitation of phosphoinositide signaling is independent of de novo phosphatidate synthesis, and that the widely used enzyme inhibitor, D-609, is probably not selective for phosphatidylcholine-specific PLC in brain slice preparations. The greater sensitivity of the CDP–DG measurement presents this assay as a reliable and possibly superior index of dopamine receptor-coupled PLC activation in intact tissues.     

Stimulation of Femoral Trabecular Bone Growth in Ovariectomized Rats by Human Parathyroid Hormone (hPTH)-(1-30)NH2

It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTH's bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH₂, which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH₂ to stimulate bone growth despite its having hPTH-(1-31)NH₂'s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH₂'s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragment's ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2–10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10–50 nmol of hPTH-(1-30)NH₂/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH₂/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH₂, [Leu²⁷]cyclo(Glu²²-Lys²⁶)-hPTH-(1-31)NH₂, hPTH-(1-34)NH₂ and [Leu²⁷]cyclo(Glu²²-Lys²⁶)-hPTH-(1-34)NH₂. These results have removed a major objection to the proposal that PTH's osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause. Received: 16 September 1998 / Accepted: 15 December 1998