Receptors Linked to Hydrolysis of Choline Phospholipids: the Role of Phospholipase D in a Putative Mechanism of Signal Transduction

We have investigated the coupling of muscarinic acetylcholine receptors (mAChR) to phospholipid hydrolysis in a human neuroblastoma cell line, LA-N-2, by measuring the formation of 3H-inositol phosphates (3H-IP) and of [3H]phosphatidylethanol ([3H]PEt) in cells prelabeled with [3H]inositol and [3H]oleic acid. The muscarinic agonist carbachol (CCh) stimulated the phospholipase C (PLC)-mediated formation of 3H-IP in a time- and dose-dependent manner (EC50 = 40-55 microM). In addition, in the presence of ethanol (170-300 mM), CCh elevated levels of [3H]PEt [which is regarded as a specific indicator of phospholipase D (PLD) activity] by three- to sixfold. The effect of CCh on PEt formation also was dose dependent (EC50 = 50 microM). Both effects of CCh were antagonized by atropine, indicating that they were mediated by mAChR. Incubation of LA-N-2 cells with the phorbol ester phorbol 12-myristate 13-acetate (PMA, 0.1 microM; 10 min) increased [3H]PEt levels by up to 10-fold. This effect was inhibited by the protein kinase C (PKC) inhibitor staurosporine (1 microM) or by pretreatment for 24 h with 0.1 microM PMA, by 74% and 65%, respectively. In contrast, the effect of CCh on PEt accumulation was attenuated by only 28% in the presence of staurosporine (1 microM). In summary, these results suggest that, in LA-N-2 neuroblastoma cells, mAChR are coupled both to phosphoinositide-specific PLC and to PLD. PKC is capable of stimulating PLD activity in these cells; however, it is not required for stimulation of the enzyme by mAChR activation.

Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Stimulation of Phospholipase D Activity in Human Neuroblastoma (LA‐N‐2) Cells by Activation of Muscarinic Acetylcholine Receptors or by Phorbol Esters: Relationship to Phosphoinositide Turnover

Sandmann

Wurtman

1991

“…What is the physiological role of PLD activated by the metabotropic receptor? PLD may be responsible for a longterm generation of diacylglycerol, the endogenous activator of PKC (reviewed by Loffelholz, 1989Loffelholz, , 1990Liscovitch, 1992). Activation of PKC through metabotropic receptor stimulation (Manzoni et al, 1990;Fukunaga et al, 1992) seems to play a crucial role in long-term aspects of cellular control such as long-term potentiation in the hippocampus (reviewed by Madison et al, 1991;Bliss and Collingridge, 1993) and long-term depression in the cerebellum (reviewed by Miller,199 1).…”

Section: Discussionmentioning

confidence: 99%

“…Very recently, 1 S,3R-1 aninocyclopentane-1,3 dicarboxylic acid (IS,3R-ACPD), a selective agonist at metabtropic glutamate receptors, was reported to stimulate phospholipase D (PLD) activity in rat hippocampal slices (Boss and Conn, 1992). PLD is the key enzyme in a novel signal transduction pathway that leads to a long-lasting, receptor-regulated formation of phosphatidic acid (PtdOH) and diacylglyml (reviewed by Uffelholz, 1989,1990, Liscovitch, 1992. However, Boss and Conn (1992) were unable to demonstrate the activation of PLD by glutamate, the putative endogenous agonist.…”

mentioning

confidence: 99%

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Holler

Cappel

Klein

et al. 1993

Self Cite

Phospholipase D (PLD) is activated by many neurotransmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) time- and concentration-dependently enhanced the formation of [3H]phosphatidylpropanol ([3H]PP) and of [3H]phosphatidic acid in the presence of 2% propanol. N-Methyl-D-aspartate and kainate (both 1 mM) caused small, but significant increases (approximately 50%), whereas alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (100 microM) was ineffective. Maximally effective concentrations of glutamate (1 mM) and of 1S,3R-ACPD (300 microM) increased the PLD activity to almost 300% of basal activity; the EC50 values were 199 and 47 microM, respectively. Glutamate receptor antagonists, such as DL-2-amino-3-phosphonopropionic acid (AP3), DL-2-amino-5-phosphonovaleric acid, and kynurenate (all 1 mM) did not inhibit the glutamate-evoked increase of PP formation. In slices of adult rats, the response to 1S,3R-ACPD was significant, but small, whereas glutamate was effective only in the presence of the glutamate uptake inhibitor L-aspartate-beta-hydroxamate. It is concluded that glutamate activates PLD in rat hippocampus through an AP3-resistant metabotropic receptor. This effect is subject to ontogenetic development, with one important factor being glutamate uptake.

“…The most well-known pathway to utilize such a mechanism includes the hydrolysis of phosphoinositides by phospholipase C. However, recent experiments have determined that many receptors are coupled to the hydrolysis of phosphatidylcholine (PC). Both phospholipase C and phospholipase D (PLD) can hydrolyze PC, but the PLD pathway probably plays the predominant role in signal transduction (for reviews, see Loffelholz, 1990; Thompson et al, 1991). This pathway converts PC into choline and phosphatidic acid (PA), which can then be converted to diacylglycerol by phosphatidate phosphohydrolase.…”

mentioning

confidence: 99%

Metabotropic Excitatory Amino Acid Receptor Activation Stimulates Phospholipase D in Hippocampal Slices

Boss

Conn

1992

Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein‐coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R‐amino‐1,3‐cyclopentanedicarboxylic acid (ACPD) and 1S,3S‐ACPD, but not the inactive isomer, 1R,3S‐ACPD, induce a concentration‐dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1S,3R‐ACPD‐induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR‐iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L‐2‐Amino‐3‐phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis‐coupled mGluRs.