Involvement of cytosolic phospholipase A(2), calcium independent phospholipase A(2) and plasmalogen selective phospholipase A(2) in neurodegenerative and neuropsychiatric conditions.

Enzymes belonging to the PLA(2) superfamily catalyze the hydrolysis of unsaturated fatty acids from the sn-2 position of glycerol moiety of neural membrane phospholipids. The PLA(2) superfamily is classified into cytosolic PLA(2) (cPLA(2)), calcium-independent PLA(2) (iPLA(2)), plasmalogen-selective PLA(2) (PlsEtn-PLA(2)) and secretory PLA(2) (sPLA(2)). PLA(2) paralogs/splice variants/isozymes are part of a complex signal transduction network that maintains cross-talk among excitatory amino acid and dopamine receptors through the generation of second messengers. Individual paralogs, splice variants and multiple forms of PLA(2) may have unique enzymatic properties, tissue and subcellular localizations and role in various physiological and pathological situations, hence tight regulation of all PLA(2) isoforms is essential for normal brain function. Quantitative RT-PCR analyses show significantly higher relative level of expression of iPLA(2) than cPLA(2) in all regions of the rat brain. Upregulation of the cPLA(2) family is involved in degradation of neural membrane phospholipids and generation of arachidonic acid-derived lipid metabolites that have been implicated in nociception, neuroinflammation, oxidative stress and neurodegeneration. In contrast, studies using a selective iPLA(2) inhibitor, bromoenol lactone, or antisense oligonucleotide indicate that iPLA(2) is an important "housekeeping" enzyme under basal conditions, whose activity is required for the prevention of vacuous chewing movements, a rodent model for tardive dyskinesia, and deficits in the prepulse inhibition of the auditory startle reflex, a common finding in schizophrenia. These studies support the view that PLA(2) activity may not only play a crucial role in neurodegeneration but depending on the isoform, could also be essential in prevention of neuropsychiatric diseases. The findings could open new doors for understanding and treatment of neurodegenerative and neuropsychiatric diseases.

Deacylation and reacylation of neural membrane glycerophospholipids.

The deacylation-reacylation cycle is an important mechanism responsible for the introduction of polyunsaturated fatty acids into neural membrane glycerophospholipids. It involves four enzymes, namely acyl-CoA synthetase, acyl-CoA hydrolase, acyl-CoA: lysophospholipid acyltransferase, and phospholipase A2. All of these enzymes have been purified and characterized from brain tissue. Under normal conditions, the stimulation of neural membrane receptors by neurotransmitters and growth factors results in the release of arachidonic acid from neural membrane glycerophospholipids. The released arachidonic acid acts as a second messenger itself. It can be further metabolized to eicosanoids, a group of second messengers involved in a variety of neurochemical functions. A lysophospholipid, the second product of reactions catalyzed by phospholipase A2, is rapidly acylated with acyl-CoA, resulting in the maintenance of the normal and essential neural membrane glycerophospholipid composition. However, under pathological situations (ischemia), the overstimulation of phospholipase A2 results in a rapid generation and accumulation of free fatty acids including arachidonic acid, eicosanoids, and lipid peroxides. This results in neural inflammation, oxidative stress, and neurodegeneration. In neural membranes, the deacylation-reacylation cycle maintains a balance between free and esterified fatty acids, resulting in low levels of arachidonic acid and lysophospholipids. This is necessary for not only normal membrane integrity and function, but also for the optimal activity of the membrane-bound enzymes, receptors, and ion channels involved in normal signal-transduction processes.

Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders.

Neural membranes contain several classes of glycerophospholipids which turnover at different rates with respect to their structure and localization in different cells and membranes. The glycerophospholipid composition of neural membranes greatly alters their functional efficacy. The length of glycerophospholipid acyl chain and the degree of saturation are important determinants of many membrane characteristics including the formation of lateral domains that are rich in polyunsaturated fatty acids. Receptor-mediated degradation of glycerophospholipids by phospholipases A(l), A(2), C, and D results in generation of second messengers such as arachidonic acid, eicosanoids, platelet activating factor and diacylglycerol. Thus, neural membrane phospholipids are a reservoir for second messengers. They are also involved in apoptosis, modulation of activities of transporters, and membrane-bound enzymes. Marked alterations in neural membrane glycerophospholipid composition have been reported to occur in neurological disorders. These alterations result in changes in membrane fluidity and permeability. These processes along with the accumulation of lipid peroxides and compromised energy metabolism may be responsible for the neurodegeneration observed in neurological disorders.

Inhibitors of intracellular phospholipase A2 activity: their neurochemical effects and therapeutical importance for neurological disorders.

Intracellular phospholipases A2 (PLA2) are a diverse group of enzymes with a growing number of members. These enzymes hydrolyze membrane phospholipids into fatty acid and lysophospholipids. These lipid products may serve as intracellular second messengers or can be further metabolized to potent inflammatory mediators, such as eicosanoids and platelet-activating factors. Several inhibitors of nonneural intracellular PLA2 have been recently discovered. However, nothing is known about their neurochemical effects, mechanism of action or toxicity in human or animal models of neurological disorders. Elevated intracellular PLA2 activities, found in neurological disorders strongly associated with inflammation and oxidative stress (ischemia, spinal cord injury, and Alzheimer's disease), can be treated with specific, potent and nontoxic inhibitors of PLA2 that can cross blood-brain barrier without harm. Currently, potent intracellular PLA2 inhibitors are not available for clinical use in human or animal models of neurological disorders, but studies on this interesting topic are beginning to emerge. The use of nonspecific intracellular PLA2 inhibitors (quinacrine, heparin, gangliosides, vitamin E) in animal model studies of neurological disorders in vivo has provided some useful information on tolerance, toxicity, and effectiveness of these compounds.

GM1 inhibits early signaling events mediated by PDGF receptor in cultured human glioma cells.

Binding of platelet-derived growth factor receptor (PDGF) to its receptor (PDGFR) activates its receptor tyrosine kinase which autophosphorylates tyrosine residues. The p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) binds to specific phosphotyrosines on PDGFR-beta and through the associated p110 catalytic subunit of PI 3-kinase catalyzes the formation of lipids that are involved in intracellular signaling. We examined if GM1 affects interactions between PDGFR-beta and specific proteins involved in PDGFR-mediated signaling. U-1242 MG cells were studied under different growth conditions using immunoprecipitation and Western Blot analysis. PDGF-stimulated the association of PDGFR-beta with p85, ras GTPase-activating protein and PLC gamma. GM1 decreased these associations in parallel with decreased tyrosine phosphorylation of PDGFR. PDGF augmented the activity of PI 3-kinase associated with PDGFR-beta, and this was attenuated by GM1. However, GM1 did not alter SH2 domains of p85. GM1 probably inhibits PDGF-induced signaling proteins with PDGFR-beta by inhibiting phosphorylation of specific tyrosines on the receptor which bind to SH2-domains on signaling proteins.

Ganglioside GM1 enhances induction by nerve growth factor of a putative dimer of TrkA.

GM1 enhances nerve growth factor (NGF)-stimulated neuritogenesis and prevents apoptotic death of PC12 cells; both may be due to enhancement of TrkA dimerization. In this study, we examined the effect of GM1 on NGF-induced TrkA dimerization in Trk-PC12 (6-24) cells. NGF increased tyrosine phosphorylation of the 140-kDa protein (TrkA monomer), and preincubation with GM1 potentiated this effect. Adding the protein cross-linker bis(sulfosuccinimidyl) suberate with NGF resulted in the appearance of two major bands (220 and 330 kDa) when probed with antibodies against TrkA or phosphotyrosine, and GM1 also enhanced this effect. We interpret the 330-kDa band as being a homodimer of TrkA. The identity of the 220-kDa band is still not certain but may consist of a posttranslationally modified form of TrkA. Our results suggest that GM1 is augmenting the effects of NGF on PC12 cells by enhancing the dimerization and activation of the TrkA receptor.

Interaction of permanently charged metoclopramide analogs with D-2 dopamine receptors.

1. The binding of permanently charged benzamides to the D-2 dopamine receptor of striatal membranes was compared with that of tertiary amine benzamides. 2. The permanently charged benzamides were able to inhibit the binding of [3H]-spiperone to striatal membranes but were less potent than the corresponding tertiary amines. 3. Removal of sodium or decreasing the pH from 7.8 to 6.2 decreased the binding of all benzamides tested, but the permanently charged analogs were affected less by these changes than the tertiary amines. 4. These results suggest that while the binding properties of the permanently charged benzamides are similar to those of the tertiary amine benzamides, there are differences in the manner in which these compounds interact with the D-2 receptor.

The interaction of ammonium, sulfonium, and sulfide analogues of metoclopramide with the dopamine D2 receptor.

A series of permanently charged ammonium and sulfonium analogues of metoclopramide as well as a permanently uncharged sulfide analogue were synthesized and evaluated for their ability to inhibit apomorphine-induced responses on mouse striatal slices. Three of the four permanently charged analogues were found to inhibit apomorphine's effects, although at higher concentrations than either metoclopramide or its dimethyl analogue. In contrast, the sulfide analogue was inactive at concentrations up to 100 microM. These findings are consistent with earlier studies of chlorpromazine and sulpiride analogues and provide further evidence that dopamine antagonists bind in their charged molecular forms to anionic sites on the D2 receptor. Further, the results of this study in conjunction with those of our earlier sulpiride study would seem to indicate that differences in the biological profiles of metoclopramide, a type 1 benzamide useful as a gastric prokinetic agent, and sulpiride, a type 2 benzamide useful for its antipsychotic effects, are not due to any appreciable differences in the binding of the basic nitrogen atom of these molecules.

Interaction of AZA analogs of chlorpromazine with the dopamine D2 receptor.

1. Permanently charged AZA analogs of chlorpromazine inhibited the binding of [3H]spiperone and antagonized the apomorphine-induced inhibition of the potassium evoked release of [3H]acetylcholine. 2. The AZA analogs were more potent in binding affinity and antagonist activity than the trimethylammonium analog of chlorpromazine but less potent than chlorpromazine. 3. These results suggest that it is possible to enhance the binding of the permanently charged trimethylammonium analog of chlorpromazine by the addition of a functional group near the quaternary nitrogen which is capable of forming a hydrogen bond with the D2 dopamine receptor. 4. However, it appears that for optimal binding, as achieved with chlorpromazine, the hydrogen-bonding proton should be on the charged nitrogen.

Synthesis and D2 dopaminergic activity of pyrrolidinium, tetrahydrothiophenium, and tetrahydrothiophene analogues of sulpiride.

All of the existing dopamine receptor models recognize the amine nitrogen of agonist and antagonist drugs as playing a crucial role in receptor interactions. However, there has been some controversy as to which molecular form of the amine, charged or uncharged, is most important in these interactions. We have synthesized and examined the biological activity of permanently charged and permanently uncharged analogues of the dopaminergic antagonist, sulpiride. Sulpiride and the permanently charged pyrrolidinium (6,7) and tetrahydrothiophenium (9) analogues were able to antagonize the inhibitory effect of apomorphine on the K+-induced release of [3H]acetylcholine from striatal slices. In contrast, the permanently uncharged tetrahydrothiophene analogue 8 was inactive at concentrations up to 100 microM. Additionally, both sulpiride and the tetrahydrothiophenium analogue were able to displace [3H]spiperone from D2 binding sites, while the tetrahydrothiophene analogue was unable to produce any significant displacement. These results are consistent with our previous observations on permanently charged chlorpromazine analogues and provide further evidence that dopaminergic antagonists bind in their charged molecular forms to anionic sites on the D2 receptor.

Effect of the inhibition of dopamine uptake on the dopamine- and dimethyldopamine-induced-inhibition of the potassium-evoked release of [3H]acetylcholine from striatal slices.

1. Dimethyldopamine was eight times more potent than dopamine in activating the D2 receptor that inhibits the potassium-evoked release of [3H]acetylcholine from striatal slices. 2. Cocaine and mazindol produced an eight-fold shift in the concentration-response curve for dopamine, but not for dimethyldopamine. 3. The IC50 of dimethyldopamine for the inhibition of [3H]dopamine uptake was thirty times greater than that for dopamine. 4. Dopamine may be less potent than dimethyldopamine at the D2 receptor because dopamine has a higher affinity for the dopamine uptake system, resulting in its rapid removal from the vicinity of the receptor.

Regulation of protein kinase C activity by various lipids.

Protein kinase C has recently attracted considerable attention because of its importance in the control of cell division, cell differentiation, and signal transduction across the cell membrane. The activity of this enzyme is altered by several lipids such as diacylglycerol, free fatty acids, lipoxins, gangliosides, and sulfatides. These lipids may interact with protein kinase C either directly or through calcium ions and produce their regulatory effect (activation or inhibition) on the activities of the enzymes phosphorylated by this kinase. These processes widen our perspective of the regulation of intercellular and intracellular communication.

Interaction of permanently uncharged dopamine analogs with the D-2 dopaminergic receptor.

The purpose of this study was to determine if structural analogs of dopamine in which the side chain nitrogen has been replaced by a permanently uncharged monomethylsulfide, monomethylselenide or sulfoxide group are capable of binding to the striatal D-2 dopamine receptor and acting as agonists at this receptor. All the permanently uncharged dopamine analogs were found to bind to the D-2 dopamine receptor as evidenced by their abilities to inhibit significantly [3H]spiperone binding to striatal homogenates. However, the inhibition of [3H]spiperone binding by the uncharged dopamine analogs was incomplete and was almost abolished by the addition of NaCl (125 mM) to the incubation medium or by the addition of dopamine or quinpirole at a concentration that that saturates the high-affinity state of the D-2 dopamine receptor. These effects of NaCl, dopamine and quinpirole suggest that the uncharged dopamine analogs bind primarily to the high-affinity state of the D-2 dopamine receptor. Whether the uncharged monomethylsulfide and sulfoxide analogs could function as dopamine agonists at the striatal D-2 dopamine receptor was assessed by determining the abilities of these compounds to inhibit the K+-evoked release of [3H]acetylcholine from striatal slices. Both the monomethylsulfide and sulfoxide analogs inhibited the K+-evoked release of [3H]acetylcholine, but this inhibitory effect does not appear to be due to the activation of the D-2 dopamine receptor since it was not reversed by the selective D-2 dopamine antagonist, sulpiride. Additionally, the uncharged monomethylsulfide and sulfoxide dopamine analogs were found to antagonize the ability of apomorphine to inhibit the K+-evoked release of [3H]acetylcholine, but this antagonistic effect does not appear to be due to the reversible blockade of the D-2 dopamine receptor since it was not reduced by increasing the concentration of apomorphine. Therefore, while the permanently uncharged analogs of dopamine appear to bind to the high-affinity state of the D-2 dopamine receptor, they are not dopamine agonists or antagonists at the striatal D-2 dopamine receptor involved in regulating the release of acetylcholine. These results suggest that a positive charge may be a requirement for the activation of the striatal D-2 dopamine receptor.

Interaction of permanently charged analogs of dopamine with the D-2 dopaminergic receptor.

Dopamine can exist in both charged and uncharged forms at physiological pH. At present it is unclear which of these forms is responsible for dopaminergic agonist activity. The purpose of this study was to determine whether permanently charged structural analogs of dopamine containing either a nitrogen, sulfur, or selenium atom in the side chain can bind to and activate the D-2 dopamine receptor. Binding to and activation of the D-2 dopamine receptor were measured by determining the abilities of the permanently charged dopamine analogs to inhibit [3H]spiperone binding to striatal homogenates and to inhibit K+-stimulated [3H]acetylcholine release from striatal slices respectively. The quaternary ammonium, dimethylsulfonium and dimethylselenonium analogs of dopamine were all found to inhibit [3H]spiperone binding to the same extent and in a manner qualitatively similar to the parent amines, dopamine and dimethyldopamine. Thus, [3H]spiperone inhibition curves for dopamine, dimethyldopamine and the permanently charged dopamine analogs were generally shallow and fit best to a two-site binding model as indicated by computer-assisted analyses. The addition of 125 mM NaCl to the incubation medium resulted in a significant decrease in the proportion of high affinity binding sites for both the permanently charged analogs and the parent amines. Similarly, the permanently charged dopamine analogs were found to maximally inhibit the K+-stimulated release of [3H]acetylcholine to the same extent as dopamine and dimethyldopamine. However, the permanently charged analogs were less potent in inhibiting both [3H]spiperone binding and K+-stimulated [3H]acetylcholine release than dopamine and dimethyldopamine. These results show that dopamine analogs possessing a permanent positive charge in the side chain can bind to and activate the D-2 dopamine receptor. The lower potencies of the permanently charged analogs in binding to and activation of the D-2 dopamine receptor suggest that, while the ability of a compound to exist in an uncharged form is not a requirement, both charged and uncharged forms of the agonist molecule appear to play a role in D-2 dopamine agonist activity.

Charged analogues of chlorpromazine as dopamine antagonists.

Chlorpromazine (1, CPZ) is a potent dopamine antagonist that has been used widely as an antipsychotic agent. Since dopaminergic antagonists, like dopaminergic agonists, exist in solution as the charged and uncharged molecular species, it is not clear which form of the amine is most important for interaction with the dopamine receptor. Previous work from our laboratory has indicated that a variety of permanently charged species could replace the amine/ammonium moiety of dopamine and retain dopamine agonist activity. This paper describes the synthesis and dopamine antagonist activity of both the trimethylammonium iodide and the dimethylsulfonium iodide analogues of chlorpromazine. The permanently uncharged methyl sulfide analogue was also synthesized; however, due to its lack of aqueous solubility, its pharmacological activity could not be evaluated. Binding of both the dimethylsulfonium iodide and the trimethylammonium iodide analogues to D-2 dopamine receptors of rat striatal tissue was observed. The observed relative order of binding was CPZ greater than CPZ sulfonium analogue greater than CPZ ammonium analogue. These compounds had a similar order of activity in antagonizing the apomorphine-induced inhibition of potassium-induced release of [3H]acetylcholine from mouse striatal slices.

Effect of permanently charged and uncharged dopaminergic agonists on the potassium-induced release of [3H]acetylcholine from striatal slices.

The effects of chemical analogs of dopamine, which are permanently charged or which lack a net positive charge, on the potassium-evoked release of [3H]acetylcholine from mouse striatal slices were studied in order to determine whether a positive charge on the dopamine agonist molecule is required to activate dopaminergic receptors. The striatal slices were first preincubated with [3H]choline, transferred to a superfusion chamber, and then superfused in physiological medium. [3H]Acetylcholine release was evoked by exposure of the slices to a high potassium medium and potential dopamine agonist drugs were added to the medium 10 min before superfusing with high potassium. A permanently charged quaternary ammonium analog and dimethylselenonium analog of dopamine inhibited the potassium-evoked release of [3H]acetylcholine, and this inhibition was antagonized by sulpiride, a dopamine receptor antagonist. However, this inhibition was not antagonized by reserpine and alpha-methyl-p-tyrosine, which was shown to completely antagonize the inhibitory effect of amphetamine, an indirectly acting amine. This suggests that the charged dopamine analogs are acting directly on dopaminergic receptors. In contrast to the permanently charged dopamine analogs, analogs of dopamine with no net positive charge produced no inhibition of the potassium-evoked [3H]acetylcholine release. These in vitro observations are in agreement with a behavioral model in which a permanently uncharged monomethylsulfide analog of dopamine was ineffective in eliciting circling behavior after its unilateral injection into the striatum of rats in which dopamine neurons were previously lesioned by the injection of 6-hydroxydopamine into the medial forebrain bundle. In contrast, under these same conditions, the intrastriatal injection of the charged quaternary ammonium or dimethylsulfonium analog of dopamine elicited intense contralateral circling. These results suggest that the charged form of a dopamine agonist molecule is required to bind to and activate the dopamine receptor regulating [3H]acetylcholine release and circling behavior.

Dopamine agonists: effects of charged and uncharged analogues of dopamine.

Dopamine, at physiological pH, may exist as either an uncharged amine or a charged ammonium species. In order to gain insight as to which species is better suited for interaction with the dopamine receptor, we have synthesized dopamine analogues in which the nitrogen atom is replaced with a neutral methyl sulfide, a neutral methyl selenide, a charged dimethylsulfonium iodide, and a charged dimethylselenonium iodide. These analogues were tested for their ability to inhibit the K+-stimulated release of [3H]acetylcholine from striatal slices. At 30 microM concentration, the charged sulfonium and selenonium salts possessed significant agonist activity while the corresponding neutral species were inactive, suggesting that a charged species is optimal for dopamine agonist activity. In addition, the methyl sulfide was converted into the corresponding sulfoxide and sulfone; however, neither of these oxidation products possessed significant activity as dopaminergic agonists.

Effects of quinine on K+ transport in heart mitochondria.

Quinine inhibits the respiration-dependent extrusion of K+ from Mg2+-depleted heart mitochondria and the passive osmotic swelling of these mitochondria in K+ and Na+ acetate at alkaline pH. These observations concur with those of Nakashima and Garlid (J. Biol. Chem. 257, 9252, 1982) using rat liver mitochondria. Quinine also inhibits the respiration-dependent contraction of heart mitochondria swollen passively in Na+ or K+ nitrate and the increment of elevated respiration associated with the extrusion of ions from these mitochondria. Quinine, at concentrations up to 0.5 mM, inhibits the respiration-dependent 42K+/K+ exchange seen in the presence of mersalyl, but higher levels of the drug produce increased membrane permeability and net K+ loss from the matrix. These results are all consistent with an inhibition of the putative mitochondrial K+/H+ antiport by quinine. However, quinine has other effects on the mitochondrial membrane, and possible alternatives to this interpretation are discussed.

K+/H+ antiport in heart mitochondria.

Heart mitochondria depleted of endogenous divalent cations by treatment with A23187 and EDTA swell in (a) K+ acetate or (b) K+ nitrate when an uncoupler is present. These mitochondria also exchange matrix 42K+ with external K+, Na+, or Li+ in a reaction that does not require respiration and is insensitive to uncouplers. Untreated control mitochondria do not swell in either medium nor do they show the passive cation exchange. Both the swelling and the exchange reactions are inhibited by Mg2+ and by quinine and other lipophilic amines. Swelling and exchange are both strongly activated at alkaline pH, and the exchange reaction is also increased markedly by hypotonic conditions. All of these properties correspond to those reported for a respiration-dependent extrusion of K+ from Mg2+-depleted mitochondria, a reaction attributed to a latent Mg2+- and H+-sensitive K+/H+ antiport. The swelling reactions are strongly inhibited by dicyclohexylcarbodiimide reacted under hypotonic conditions, but the exchange reaction is not sensitive to this reagent. Heart mitochondria depleted of Mg2+ show marked increases in their permeability to H+, to anions, and possibly to cations, and the permeability to each of these components is further increased at alkaline pH. This generalized increase in membrane permeability makes it likely that K+/H+ antiport is not the only pathway available for K+ movement in these mitochondria. It is concluded that the swelling, 42K+ exchange, and K+ extrusion data are all consistent with the presence of the putative K+/H+ antiport but that definitive evidence for the participation of such a component in these reactions is still lacking.