Stephen L. Yates

Phospholipid hydrolysis and loss of membrane integrity following treatment of rat brain synaptosomes with β-bungarotoxin, notexin, and Naja naja atra and Naja nigricollis phospholipase A2

The effects of the phospholipase A2 (PLA2) toxins, beta-bungarotoxin and notexin, and the PLA2 enzymes from Naja naja atra and Naja nigricollis snake venoms on the plasma membrane integrity of synaptosomes were examined. Synaptosomes were isolated from rat brain cerebral cortex, corpus striatum and hippocampus. Osmotic activity, lactate dehydrogenase leakage, and leakage of 2-deoxy-D-(1-3H)-glucose-6-phosphate were monitored (37 degrees C, 10-120 min) following incubation with 0.5, 5 and 50 nM concentrations of toxins and enzymes. Damage to the synaptosomal plasma membrane was time and concentration but not tissue dependent. The potencies of the treatments were as follows: N. n. atra PLA2 greater than or equal to N. nigricollis PLA2 greater than notexin greater than beta-bungarotoxin. Chelation of Ca2+ with 5 mM EDTA completely inhibited plasma membrane disruption caused by beta-bungarotoxin and N. n. atra PLA2. One mg/ml of bovine serum albumin also blocked the disruptive action of N. n. atra PLA2, while 8 mg/ml was required to antagonize beta-bungarotoxin. A correlation between phospholipid hydrolysis and loss of membrane integrity was also observed. The generation of phospholipid hydrolytic products may be critical in the permeabilization of synaptic plasma membranes by these toxins and enzymes, however, they do not explain the presynaptic specificity and potency of beta-bungarotoxin and notexin.

Effects of selected histamine H3 receptor antagonists on tele-methylhistamine levels in rat cerebral cortex.

The H3 antagonist thioperamide is thought to act on brain H3 autoreceptors to increase both the release and metabolism of neuronal histamine (HA). Our studies investigated the effects of several new brain-penetrating H3 antagonists on rat cerebral cortical levels of the HA metabolite tele-methylhistamine (t-MH). Animals were pretreated with H3 antagonists (0.3 to 30 mg/kg; 1-4 hr; i.p.) in the presence or absence of the monoamine oxidase inhibitor pargyline to prevent metabolism of t-MH. Cortical t-MH levels were measured by both radioimmunoassay (RIA) and gas chromatography-mass spectrometry (GC-MS). Pargyline (60 mg/kg; 1 hr; i.p.) produced an approximately 2-fold increase in t-MH levels as measured by either GC-MS or RIA. Thioperamide (+/- pargyline) increased t-MH levels as measured by both GC-MS and RIA. In contrast, neither 5-cyclohexyl-1-(4-imidazol-4-ylpiperidyl)pentan-1-one (GT-2016) (+/- pargyline), 4-(6-cyclohexylhex-cis-3-enyl)imidazole (GT-2227) (+/- pargyline), nor clobenpropit (minus pargyline) increased t-MH levels as measured by GC-MS. A good agreement was found between t-MH levels as determined by either RIA or GC-MS except after treatment with GT-2016, which increased apparent t-MH brain levels according to the former but not the latter method. Subsequent studies suggest the in vivo formation of a GT-2016 metabolite, which can cross-react in the t-MH RIA. Although all H3 receptor antagonists studied to date seem capable of enhancing brain HA release, only thioperamide presently was found to enhance cortical t-MH levels. Thus, H3 receptor antagonists may differentially affect HA release and turnover, and brain t-MH levels may not be reliable predictors of H3 agonist, partial agonist, or antagonist in vivo activity.

Inhibitory effect of EDTA · Ca2+ on the hydrolysis of synaptosomal phospholipids by phospholipase A2 toxins and enzymes

Phospholipases A2 (PLA2) are Ca2(+)-dependent enzymes that are inhibited by EDTA; this inhibition would be expected to be reversed by restoring the Ca2+ concentration. By examining the hydrolysis of synaptosomal phospholipids by PLA2 enzymes, Naja naja atra and Naja nigricollis, and by toxins with PLA2 activity, beta-bungarotoxin (beta-BuTX) and notexin, we demonstrated a novel inhibitory action of EDTA manifested in the presence of excess Ca2+. We postulate the formation of an EDTA.Ca2+ complex which inhibits PLA2 activity in a concentration-dependent manner. Synaptosomes in which phospholipids are hydrolyzed by PLA2 have membranal damage expressed by increased acetylcholine (ACh) release and decreased osmotic activity. Addition of EDTA.Ca2+, which inhibits phospholipid hydrolysis, also reversed the PLA2 effect on ACh release, but not its effect on osmotic activity. The inhibition of PLA2 was observed on membranal phospholipids as well as on an artificial substrate of phospholipid-Triton mixed micelles. Moreover, we found that another enzyme, lactate dehydrogenase, was also inhibited. Our results indicate a non-specific inhibition exerted on the enzyme rather than on the substrate.

Comparative effects of phospholipase A2 neurotoxins and enzymes on membrane potential and Na+K+ ATPase activity of rat brain synaptosomes

Beta-Bungarotoxin (beta-BuTX) and notexin are phospholipase A2 (PLA2) neurotoxins which cause an irreversible blockade of neurotransmitter release through specific and potent effects at the presynaptic nerve terminal; however, their mechanism of action is uncertain. We examined the effects of beta-BuTX and notexin on Na+/K+ ATPase activity using Sprague-Dawley rat brain synaptosomes in order to determine if alterations in activity might modulate neurotoxin-induced depolarization. Treatment of synaptosomes with 0.05 to 5 nM beta-BuTX, notexin, and Naja naja atra and Naja nigricollis PLA2 (PLA2 enzymes without selective presynaptic actions) caused a dose-dependent depolarization of synaptosomes with no differences being observed between the effects of the PLA2 neurotoxins and enzymes. N. nigricollis PLA2 (0.5 nM; 20 min) slightly stimulated Na+/K+ ATPase activity while beta-BuTX and notexin (0.5 nM: 10 and 20 min) were without effect. With 50 nM concentrations beta-BuTX and notexin stimulated Na+/K+ ATPase activity, while N. nigricollis and N. n. atra PLA2 inhibited activity. The effects on membrane potential and Na+/K+ ATPase were antagonized or blocked by EDTA (10 mM) and bovine serum albumin (1 mg/ml), suggesting that PLA2 enzymatic activity is essential for their effects on membrane potential and Na+/K+ ATPase activity. Following neurotoxin and enzyme pretreatment, we found a biphasic correlation between synaptosomal free fatty acid (FFA) levels and Na+/K+ ATPase activity, where Na+/K+ ATPase is stimulated by low levels of FFA (0.13 to 0.22 mumol/mg protein) and antagonized by FFA levels in excess of 0.34 mumol/mg protein. In contrast there was a linear correlation between the extent of FFA production and membrane depolarization. We propose that the presynaptic depolarizing actions of beta-BuTX and notexin are not mediated through modulation of Na+/K+ ATPase activity and that the changes observed in ATPase activity and possibly membrane potential are directly due to PLA2 enzymatic activity and the production of FFA.