title: Diacylglycerol-mediated regulation of Aplysia bag cell neuron excitability requires protein kinase C.
abstract: In Aplysia, reproduction is initiated by the bag cell neurons and a prolonged period of enhanced excitability known as the afterdischarge. Phosphoinositide turnover is upregulated during the afterdischarge resulting in the hydrolysis of phosphatidylinositol-4,5-bisphosphate by phospholipase C (PLC) and the release of diacylglycerol (DAG) and inositol trisphosphate (IP3 ). In whole-cell voltage-clamped cultured bag cell neurons, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic DAG analogue, activates a dose-dependent, transient, inward current (IOAG ) that is enhanced by IP3 , mimicked by PLC activation and dependent on basal protein kinase C (PKC) activity. OAG depolarizes bag cell neurons and triggers action potential firing in culture, and prolongs electrically stimulated afterdischarges in intact bag cell neuron clusters ex vivo. Although PKC alone cannot activate the current, it is required for IOAG ; this is the first description of required obligate PKC activity working in concert with PLC, DAG and IP3 to maintain the depolarization required for prolonged excitability in Aplysia reproduction.
  
  
  
  Following synaptic input, the bag cell neurons of Aplysia undergo a long-term afterdischarge of action potentials to secrete egg-laying hormone and initiate reproduction. Early in the afterdischarge, phospholipase C (PLC) hydrolyses phosphatidylinositol-4,5-bisphosphate into inositol trisphosphate (IP3 ) and diacylglycerol (DAG). In Aplysia, little is known about the action of DAG, or any interaction with IP3 ; thus, we examined the effects of a synthetic DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol (OAG), on whole-cell voltage-clamped cultured bag cell neurons. OAG induced a large, prolonged, Ca(2+) -permeable, concentration-dependent inward current (IOAG ) that reversed at ∼-20 mV and was enhanced by intracellular IP3 . A similar current was evoked by either another DAG analogue, 1,2-dioctanoyl-sn-glycerol (DOG), or activating PLC with N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide (m-3M3FBS). IOAG was reduced by the general cation channel blockers Gd(3+) or flufenamic acid. Work in other systems indicated that OAG activates channels independently of protein kinase C (PKC); however, we found pretreating bag cell neurons with any of the PKC inhibitors bisindolylmaleimide, sphinganine, or H7, attenuated IOAG . However, stimulating PKC with phorbol 12-myristate 13-acetate (PMA) did not evoke current or enhance IOAG ; moreover, unlike PMA, OAG failed to trigger PKC, as confirmed by an independent bioassay. Finally, OAG or m-3M3FBS depolarized cultured neurons, and while OAG did not provoke afterdischarges from bag cell neurons in the nervous system, it did double the duration of synaptically elicited afterdischarges. To our knowledge, this is the first report of obligate PKC activity for IOAG gating. An interaction between phosphoinositol metabolites and PKC could control the cation channel to influence afterdischarge duration.
authors.affiliation: Department of Biomedical and Molecular Sciences, Physiology Graduate Program, Queen's University, Kingston, ON, Canada, K7L 3N6.
authors.forename: Raymond M
authors.initials: RM
authors.lastname: Sturgeon
authors.affiliation: Department of Biomedical and Molecular Sciences, Physiology Graduate Program, Queen's University, Kingston, ON, Canada, K7L 3N6. magoski@queensu.ca.
authors.forename: Neil S
authors.initials: NS
authors.lastname: Magoski
mesh_terms: D000818:Animals; D001048:Aplysia; D002478:Cells, Cultured; D004075:Diglycerides / Q000494:pharmacology*; D004558:Electric Stimulation; D009474:Neurons / Q000187:drug effects* / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology*; D013449:Sulfonamides / Q000494:pharmacology; D010738:Type C Phospholipases / Q000502:physiology*; D000096926:Benzenesulfonamides
chemical_list: D004075:Diglycerides; D011493:Protein Kinase C; D013449:Sulfonamides; D010738:Type C Phospholipases; C473783:2,4,6-trimethyl-N-(meta-3-trifluoromethylphenyl)benzenesulfonamide; D000096926:Benzenesulfonamides; C044295:1,2-dioctanoylglycerol; C038237:1-oleoyl-2-acetylglycerol