Proc. Nati. Acad. Sci. USA Vol. 83, pp. 3003-3007, May 1986 Neurobiology Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: Stimulatory effects of agents that enhance influx of sodium ions (inositol phosphates/pumiliotoxin/batrachotoxin/tetrodotoxin/saidtoxin) FABIAN GUSOVSKY, ELIZABETH B. HOLLINGSWORTH, AND JOHN W. DALY Laboratory of Bioorganic Chemistry, Building 4, Room 212, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Bernhard Witkop, December 2, 1985 ABSTRACT Norepinephrine and carbamoylcholine stim- might be the basis for the biochemical and pharmacological ulate accumulation of [33H]inositol phosphates from [3H]- activity of this alkaloid. The finding also suggested that inositol-labeled guinea pig cerebral cortical synaptoneuro- nervous activity involving sodium channels might have a somes through interaction with a1-adrenergic and muscarinic regulatory input to the systems involved in phosphatidylino- receptors, respectively. In addition to such agonists, a variety sitol turnover. Sodium channel activity does appear to of natural products that affect voltage-dependent sodium provide another stimulatory input to phosphatidylinositol channels can markedly stimulate accumulation of [3H]inositol systems, since a variety of agents that enhance sodium phosphates. These include (a) alkaloids that activate sodium channel function have now been shown to stimulate phos- channels, such as batrachotoxin, veratridine, and aconitine; (ii) phatidylinositol turnover in brain preparations. peptide toxins that alter activation or slow inactivation of sodium channels, such as various scorpion toxins from Leiurus, MATERIALS AND METHODS Centruroides, and Tityus species; and (ii) agents that cause repetitive fring of sodium channel-dependent action poten- Materials. [3H]Inositol (14 Ci/mmol; 1 Ci = 37 GBq) was tials, such as pyrethroids and pumiliotoxin B. Ouabain, an purchased from Amersham. Batrachotoxin was isolated from agent that will increase accumulation of internal sodium by the skin of the dart poison frog Phyllobates terribilis (15). inhibition of Na+,K+-ATPase, also stimulates formation of Pumiliotoxin B was isolated from the skin of the poison frog [3H]inositol phosphates, as does monensin, a sodium iono- Dendrobates pumilio (16). Scorpion venoms (Leiurus phore. Tetrodotoxin and saxitoxin, specific blockers ofvoltage- quinquestriatus, Tityus serrulatus), carbamoylcholine, nor- dependent sodium channels, prevent or reduce the stimulatory epinephrine, tetrodotoxin, atropine sulfate, veratridine, effects of sodium channel agents and ouabain on phosphati- monensin, and aconitine were from Sigma. AG 1-X8 (100-200 dylinositol turnover, while having lesser or no effect, respec- mesh, formate form) anion-exchange resin was from Bio- tively, on receptor-mediated or monensin-mediated stimula- Rad. Prazosin was provided by Pfizer (Groton, CT). Scorpion tion. Removal of extracellular sodium ions markedly reduces (Leiurus) toxin (an a-scorpion toxin) was kindly provided by stimulatory effects of sodium channel agents, while removal of W. Catterall (University of Washington, Seattle). Another extracellular calcium ions with EGTA blocks both receptor- scorpion (Centruroides suffusus) toxin (toxin II, /-scorpion mediated and sodium channel agent-mediated phosphatidyl- toxin) was kindly provided by F. Couraud (Institut National inositol turnover. The results provide evidence for a hitherto de la Sante et de la Recherche Mddicale, Marseille, France). unsuspected messenger role for sodium ions in excitable tissue, The pyrethroids deltamethrin and permethrin were kindly whereby neuronal activity and the resultant influx of sodium provided by G. Brown (University of Alabama, Birming- will cause activation of phospholipase systems involved in ham). hydrolysis of phosphatidylinositols, thereby generating two Preparation of Synaptoneurosomes. Male Hartley guinea second messengers, (') the inositol phosphates, which mobilize pigs (175-220 g) were decapitated and the brains were rapidly calcium from internal stores, and (ii) the diacylglycerols, which removed. The cerebral cortex was dissected out and activate protein kinase C. synaptoneurosomes were obtained according to Hollings- worth et al. (17). Briefly, the cortex of one brain was Biogenic amines, acetylcholine analogs, and certain peptides homogenized in 7-10 vol of Krebs-Henseleit buffer in a stimulate phosphatidylinositol turnover in brain tissue glass-glass homogenizer (five strokes). When the nonffitered through interaction with specific receptors (refs. 1-10 and preparation was used, the suspension was centrifuged at this references therein). The mechanism(s) involved in receptor- point at 1000 x g for 10 min, the supernatant was decanted, mediated stimulation ofphosphatidylinositol turnover are not and the pellet was reconstituted in an appropriate volume of well understood. The products of phosphatidylinositol turn- buffer. When a filtered preparation was used, the over, namely inositol phosphates and diacylglycerides, ap- homogenate was diluted with 35 ml offresh buffer and filtered parently serve as "second messengers," with the inositol first through two layers ofnylon material (100 mesh) and then phosphates eliciting mobilization of internal calcium and the with pressure through 10-,um-pore Millipore filters (LCWP- diacylglycerides activating protein kinase C (11). An alkaloid, 047). The filtrate was centrifuged at 1000 x g for 10 min, the pumiliotoxin B, which causes repetitive firing of neurons and supernatant was decanted, and the pellet was resuspended in enhances excitation contraction coupling (12, 13), was found an appropriate volume of buffer. The synaptoneurosome to stimulate phosphatidylinositol turnover (14), suggesting preparation contains many synaptosomes with attached that generation of diacylglycerides and inositol phosphates resealed postsynaptic entities (neurosomes) (17) and is prov- ing to be a useful, readily obtained, brain preparation for a The publication costs of this article were defrayed in part by page charge variety ofbiochemical studies on cyclic AMP generation (ref. payment. This article must therefore be hereby marked "advertisement" 17 and references therein), phosphatidylinositol breakdown in accordance with 18 U.S.C. §1734 solely to indicate this fact. (9, 14), and ion fluxes (18, 19). 3003 Downloaded by guest on September 26, 2021 3004 Neurobiology: Gusovsky et al. Proc. Natl. Acad. Sci. USA 83 (1986) Phosphatidylinositol Turnover. The nonfiltered pellet from RESULTS one guinea pig brain or the combined filtered pellet from two guinea pigs was resuspended in 20 ml of Krebs-Henseleit Carbamoylcholine and norepinephrine stimulate phosphati- buffer. Similar results were obtained with nonfiltered and dylinositol turnover in [3H]inositol-labeled guinea pig cere- filtered pellets. To the suspension was added 270 ,Ci of bral cortical synaptoneurosomes (Table 1). The effects of are completely blocked by a muscarinic [3H]inositol (1 ,M), and the mixture was incubated for 60mm carbamoylcholine at 37TC with continuous gassing with 02/CO2 (95:5, vol/vol). antagonist, atropine, while effects of norepinephrine are blocked by a specific a1-adrenergic antagonist, After centrifugation at 1000 x g for 10 min and decantation completely of the supernatant, the labeled synaptoneurosomes were prazosin (data not shown). Tetrodotoxin caused a 20-35% reconstituted in 11-14 ml of fresh buffer containing 10 mM inhibition of these receptor-mediated responses (Table 1). LiCl so that 320-,l aliquots would contain approximately 1 Saxitoxin (0.5 ,M) in one experiment caused 40% inhibition mg of protein. Aliquots were transferred to polypropylene of the carbamoylcholine response and a 9% inhibition of the tubes (Falcon 2063) and, after 10 min at room temperature, norepinephtine response. Such inhibitions suggest that re- agents in buffer or buffer alone (20,ul) was added. The tubes ceptor-mediated stimulation of phosphatidylinositol break- were then gassed briefly with 02/CO2 (95:5), capped, and down in brain preparations involves to some extent influx of placed in a water bath at 37°C. Unless stated otherwise sodium ions through voltage-dependent sodium channels. incubations were for 90 min. A variety of agents that are known to affect the state or The analysis of [3H]inositol metabolites was as described function of voltage-dependent sodium channels enhance by Berridge et al. (1, 20). In brain preparations, the major accumulation of [3H]inositol phosphate in synaptoneuro- product ofphosphatidylinositol breakdown in the presence of somes (Table 1). The time courses for responses to a receptor lithium ions is inositol phosphate, with inositol bis- and agonist, carbamnoylcholine, to scorpion (Leiurus) venom, and trisphosphates being very minor (data not shown; see also to pumiliotoxin B are shown in Fig. 1. The concentration refs. 1-10). In the present study, total accumulation of dependency of the effect of purified scorpion (Leiurus) toxin [3H]inositol phosphates in inositol-labeled synaptoneuro- on phosphatidylinositol breakdown is shown in Fig. 2. The somes has been used to monitor phosphatidylinositol turn- concentration of 2 nM for half-maximal effect is commensu- over. Briefly, the tubes were centrifuged for 5 min at 1000 x rate with the potency of scorpion toxin in enhancing sodium g and the supernatant was discarded. The pellets were fluxes and allosterically