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Since the discovery in 1953 by Hokin and Hokin of an increased 32 P-labelling of phospholipids upon stimulation of muscarinic receptors with acetylcholine, there have been great advances in our understanding of the way in which receptors are coupled to the phosphoinositide cycle and the way in which the phosphoinositide derived second messengers inositol 1,4,5-trisphosphate and diacylglycerol mediate a number of cellular processes. In recent years, there has been considerable interest in studying the modulation of such receptor-mediated inositol phospholipid breakdown responses in the brain, both in vitro by feedback, receptor crosstalk and desensitization mechanisms as well as by modulatory agents, such as metal ions and ex vivo following a number of surgical and pharmacological treatments. These studies are reviewed in the present article.

The calcium and potassium ion dependencyof the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 μM) potentiated the response to carbachol at 6 mM K + when Ca 2+ -free, but not when 2.52 mM Ca 2+ assay buffer was used. In Ca 2+ -free buffer, verapamil (10 μM) inhibited the response to carbachol at both 6 and 18 mM K + , but higher concentrations (30–300 μM) were needed when 2.52 mM Ca 2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [ 3 H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 μM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K + at 1.3 mM Ca 2+ , but was without effect on the basal rate at other K + and Ca 2+ concentrations. In the presence of NMDA (100 μM) or quisqualate (100 μM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K + and Ca 2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.

The effects of ouabain and monensin upon basal and carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms have been investigated. Basal inositol phospholipid breakdown was increased by both compounds at both 6 and 18 mM K+. Enhancement of the carbachol reponse at 6 mM, but not at 18 mM K+, was found with high concentrations of ouabain. On the other hand, monensin blocked the response to carbachol. Monensin also inhibited the specific binding of [3H]pirenzepine to cerebral cortical membranes, but this was found only at concentrations higher than required to affect the basal and carbachol-stimulated inositol phospholipid breakdown responses. Quabain did not affect [3H]pirenzepine binding at any of the concentrations tested (6–600 μM). It is concluded that agents that increase the intracellular sodium ion concentration affect the inositol phospholipid breakdown response to carbachol, but that the modulation can be both to potentiate and to inhibit the response.

The endocannabinoid system consists of the cannabinoid (CB) receptors, CB(1) and CB(2), the endogenous ligands anandamide (AEA, arachidonoylethanolamide) and 2-arachidonoylglycerol (2-AG), and their synthetic and metabolic machinery. The use of cannabis has been described in classical and recent literature for the treatment of pain, but the potential for psychotropic effects as a result of the activation of central CB(1) receptors places a limitation upon its use. There are, however, a number of modern approaches being undertaken to circumvent this problem, and this review represents a concise summary of these approaches, with a particular emphasis upon CB(2) receptor agonists. Selective CB(2) agonists and peripherally restricted CB(1) or CB(1)/CB(2) dual agonists are being developed for the treatment of inflammatory and neuropathic pain, as they demonstrate efficacy in a range of pain models. CB(2) receptors were originally described as being restricted to cells of immune origin, but there is evidence for their expression in human primary sensory neurons, and increased levels of CB(2) receptors reported in human peripheral nerves have been seen after injury, particularly in painful neuromas. CB(2) receptor agonists produce antinociceptive effects in models of inflammatory and nociceptive pain, and in some cases these effects involve activation of the opioid system. In addition, CB receptor agonists enhance the effect of mu-opioid receptor agonists in a variety of models of analgesia, and combinations of cannabinoids and opioids may produce synergistic effects. Antinociceptive effects of compounds blocking the metabolism of anandamide have been reported, particularly in models of inflammatory pain. There is also evidence that such compounds increase the analgesic effect of non-steroidal anti-inflammatory drugs (NSAIDs), raising the possibility that a combination of suitable agents could, by reducing the NSAID dose needed, provide an efficacious treatment strategy, while minimizing the potential for NSAID-induced gastrointestinal and cardiovascular disturbances. Other potential "partners" for endocannabinoid modulatory agents include alpha(2)-adrenoceptor modulators, peroxisome proliferator-activated receptor alpha agonists and TRPV1 antagonists. An extension of the polypharmacological approach is to combine the desired pharmacological properties of the treatment within a single molecule. Hopefully, these approaches will yield novel analgesics that do not produce the psychotropic effects that limit the medicinal use of cannabis.

In this communication, a new approach to enhancing the efficacy of liquid−liquid anion exchange is demonstrated. It involves the concurrent use of appropriately chosen hydrogen-bond-donating (HBD) anion receptors in combination with a traditional quaternary ammonium extractant. The fluorinated calixpyrroles 1 and 2 and the tetraamide macrocycle 4 were found to be particularly effective receptors. Specifically, their use allowed the extraction of sulfate by tricaprylmethylammonium nitrate to be effected in the presence of excess nitrate. As such, the present work provides a rare demonstration of overcoming the Hofmeister bias in a competitive environment and the first to the authors’ knowledge wherein this difficult-to-achieve objective is attained using a neutral HBD-based anion binding agent under conditions of solvent extraction.

A series of para-substituted phenolic N-alkyl carbamates were evaluated for their FAAH and MGL inhibitory activities. The compounds were generally selective for FAAH, with IC(50) values in the nM range, whereas inhibition of MGL required concentrations three orders of magnitude higher. The most potent compounds, dodecylcarbamic acid 4-(4,5-dihydrothiazol-2-yl)phenyl (12) and 4-(1,2,3-thiadiazol-4-yl)phenyl (26) esters, inhibited FAAH and MGL with IC(50) values at the low-nanomolar (IC(50)s; 0.0063 and 0.012 microM) and the low-micromolar ranges (IC(50)s; 2.1 and 1.0 microM), respectively. Compound 26 also inhibited both FAAH-dependent AEA uptake and AEA hydrolysis (IC(50); 0.082 microM) by intact RBL2H3 cells, and could also reduce 2-AG hydrolysis by these cells at concentrations >or=0.030 microM.

The actions of anandamide and 2-arachidonoylglycerol are terminated by cellular uptake followed by metabolism. In the case of anandamide, the uptake process was originally suggested to be achieved by a process of facilitated diffusion, but the mechanism(s) involved are a matter of controversy at present. The main hydrolytic enzyme for anandamide is fatty acid amide hydrolase, and inhibitors of this enzyme have been found to have beneficial effects in animal models of inflam- matory pain, inflammation, anxiety and depression. Anandamide is also a substrate for cyclooxygenase-2 and lipoxygenases, and the cyclooxygenase-derived products, the "prostamides" have biological actions of their own. 2-Arachidonoylglycerol can be metabolized by a range of enzymes, including cyclooxygenase-2, monoacylg- lycerol lipase and fatty acid amide hydrolase. In the brain, monoacylglycerol lipase is probably the most important of these enzymes. However, no selective inhibitors of this enzyme are presently available with which to establish the potential of this enzyme as a target for drug development.

The effects of sodium fluoride upon basal and agonist-stimulated inositol phospholipid breakdown have been investigated in rat brain miniprisms. NaF concentration dependently increased basal inositol phospholipid breakdown, with a maximum effect being seen at 20 mM. NaF reduced the inositol phospholipid breakdown responses to stimulation by carbachol, noradrenaline, serotonin and quisqualate, but not to the stimulation produced by raising the assay [K+] from 6 to 18 mM. More detailed study demonstrated NaF to have a 'levelling' effect, reducing all InsP/(Lipid + InsP) values greater than 0.15 (i.e. produced by carbachol at raised [K+], noradrenaline and by 50 mM K+) to about this value. Time-course experiments indicated that NaF treatment reduced the rate of carbachol-stimulated inositol phospholipid breakdown up to this InsP/(Lipid + InsP) level and thereafter blocked further breakdown. Inhibitory effects upon carbachol-stimulated inositol phospholipid breakdown were not seen with forskolin, sodium nitroprusside or 8BrcGMP. Under conditions where there is no de novo synthesis of phosphoinositides from [3H]myo-inositol, NaF reduced the total Lipid + InsP labelling by about 20%. NaF in addition inhibits the activity of Ins(1,4)P2-phosphatase in cerebral cortical homogenates. It is concluded that fluoride ions inhibit agonist-stimulated inositol phospholipid breakdown via actions not only on G-proteins but also on phosphoinositide-specific phospholipase C substrate availability.

The hydrophilic beta-adrenoceptor ligand (-)-[3H]CGP-12177 binds to intact C6 cells with a high affinity (KD approximately 0.1 nM) and with a high degree of specificity. The binding was inhibited by DL-propranolol (Ki approximately 1 nM). Treatment of cells cultured in Dulbecco's modified Eagle medium (DMEM) without fetal calf serum for 4 days with desipramine reduced the (-)-[3H]CGP-12177 specific binding in a concentration-dependent manner, a reduction from 127 to 102 fmol/mg of protein being found at a ligand concentration of 1 nM after treatment with 10 microM desipramine. Lesser effects were seen after treatment for 1 day. A similar result was found with maprotiline, and reductions in specific binding were seen after 4 days of treatment with amitriptyline, iprindole, and citalopram. The reduction in binding-site density (measured per milligram of protein to compensate for variability in cell density per well), however, was paralleled in all cases by a reduction in the rate of cell proliferation. When C6 glioma cells were cultured in Ham's medium without fetal calf serum during the antidepressant treatment period, a higher specific binding was observed than for the DMEM-cultured cells, and 10 microM desipramine was without effect on either the (-)-[3H]CGP-12177 specific binding or cell proliferation. It is concluded that the effects of the antidepressants tested upon the density of (-)-[3H]CGP-12177 specific binding sites in intact C6 cells may be secondary to the toxicity of the compounds under the conditions used.

There is evidence in the literature that the nonsteroidal anti-inflammatory drugs indomethacin and ibuprofen can interact with the cannabinoid system both in vitro and in vivo. In the present study, a series of analogues of ibuprofen and indomethacin have been investigated with respect to their ability to inhibit fatty acid amide hydrolase, the enzyme responsible for the hydrolysis of the endogenous cannabinoid anandamide. Of the fourteen compounds tested, the 6-methyl-pyridin-2-yl analogue of ibuprofen ("ibu-am5") was selected for further study. This compound inhibited rat brain anandamide hydrolysis in a non-competitive manner, with IC50 values of 4.7 and 2.5 microM being found at pH 6 and 8, respectively. By comparison, the IC50 values for ibuprofen were 130 and 750 microM at pH 6 and 8, respectively. There was no measurable N-acylethanolamine hydrolyzing acid amidase activity in rat brain membrane preparations. In intact C6 glioma cells, ibu-am5 inhibited the hydrolysis of anandamide with an IC50 value of 1.2 microM. There was little difference in the potencies of ibu-am5 and ibuprofen towards cyclooxygenase-1 and -2 enzymes, and neither compound inhibited the activity of monoacylglycerol lipase. Ibu-am5 inhibited the binding of [3H]-CP55,940 to rat brain CB1 and human CB2 cannabinoid receptors more potently than ibuprofen, but the increase in potency was less than the corresponding increase in potency seen for inhibition of FAAH activity. It is concluded that ibu-am5 is an analogue of ibuprofen with a greater potency towards fatty acid amide hydrolase but with a similar cyclooxygenase inhibitory profile, and may be useful for the study of the therapeutic potential of combined fatty acid amide hydrolase-cyclooxygenase inhibitors.

Publisher Summary This chapter discusses supramolecular chemistry of environmentally relevant anions. The ultimate goal of the studies on the basic chemical aspects of anion receptor design of functional pH-independent systems is to target selective binding of anions of environmental significance. In applications-oriented aspects of the research on the design of separations, strategies for selective and efficient removal of targeted anions have been a major long-term goal. The chapter discusses (1) some of the first synthetic sulfate-selective anion-binding agents; (2) simple, structure-based methods for modifying the intrinsic anion selectivity of a given class of anion receptors; and (3) the first system capable of extracting sulfate anion from acidic, nitrate-containing aqueous media. These initial findings point the way to more effective systems involving receptors with higher selectivity, binding strength, and solubility. The evolution of anion-binding ligands from simple ammonium-based systems through mixed amide/amine and thioamide/amine receptors is reviewed. Thus, the emphasis is largely on synthetic design strategy carried out in the Bowman–James group.

It might have seemed unlikely that something as deceptively straightforward as the cellular uptake of two arachidonic acid derivatives would cause so much controversy, but that is exactly the way things have turned out for the endocannabinoids anandamide and 2-arachidonoylglycerol since the first detailed description of their uptake [1]. The original debate concerned the existence (or not) of a designated membrane transporter protein, because a simple diffusion model followed by intracellular transport can explain current data as well as a model invoking an as-yet-unidentified membrane transporter [2].

An increasing number of signal transduction disturbances have been reported in Alzheimer's disease. These changes are not restricted to histopathologically changed brain areas but are seen also in peripheral tissues. One of the most severe disturbances is a loss of calcium-mobilizing intracellular inositol(1,4,5)-trisphosphate receptors in Alzheimer cerebellar and cortical tissues. In the present study, the binding of [3H]inositol(1,4,5)trisphosphate ([3H]Ins(1,4,5)P3) to the calcium-mobilizing inositol(1,4,5)trisphosphate receptors in platelet membranes from eight Alzheimer's disease patients and eight control subjects were investigated to determine its possible role as a biological marker in Alzheimer's disease. It was found that there were no significant difference in [3H]Ins(1,4,5)P3 binding with respect to the number of sites measured at different protein concentrations or to the sensitivity of the binding to inhibition by nonradioactive Ins(1,4,5)P3 between Alzheimer disease platelets and controls. It is concluded that inositol(1,4,5)trisphosphate receptor levels are preserved in platelets from patients with Alzheimer's disease.

In the present study, the abilities of cyclooxygenated derivatives of 1-arachidonoylglycerol and related compounds to prevent the metabolism of [3H]2-oleoylglycerol and [3H]anandamide by cytosolic and membrane fractions, respectively, have been investigated. For each compound, nine concentrations (range 0.2-100 microM) were tested. 1-Arachidonoylglycerol inhibited the hydrolysis of [3H]2-oleoylglycerol with a pI50 value of 5.17+/-0.04 (maximum attainable inhibition 88%). In contrast, the 1-glyceryl esters of prostaglandin D2, E2 and F2alpha were very weak inhibitors of this hydrolysis. Similarly, prostaglandin D2, prostaglandin D2 ethanolamide and prostaglandin D2 serinol amide produced20% inhibition of [3H]2-oleoylglycerol metabolism at any concentration tested, in contrast to previous data with arachidonic acid, anandamide and arachidonoyl serinol which are all able to inhibit metabolism of this substrate under the assay conditions used here. A similar pattern was seen for all the compounds with respect to the inhibition of [3H]anandamide hydrolysis by the membrane fractions. Thus, cyclooxygenation of the arachidonoyl side chain greatly reduces the ability of 1-arachidonoylglycerol and related compounds to inhibit the hydrolysis of [3H]2-oleoylglycerol and [3H]anandamide.

There is considerable controversy at present concerning the mechanisms responsible for the cellular uptake of anandamide. One particular issue concerns whether fatty acid-free bovine serum albumin should be used in the assays, it having been argued that such a presence effectively prevents the specific uptake of anandamide. In the present study, it has been demonstrated that in the presence of a low (0.1%, w/v) concentration of fatty acid-free bovine serum albumin, a temperature-dependent and saturable (K(m) approximately 1 microM) uptake of anandamide into P19 embryonic carcinoma cells can be demonstrated using an incubation time of 4 min. Under these conditions, the uptake of anandamide at 4 degrees C is low at a substrate concentration of 100 nM. The uptake at 37 degrees C was not significantly reduced following treatment of the cells with either methyl-beta-cyclodextrin (50 microM) or mevinolin (1 microM), but was reduced by the FAAH inhibitor URB597 (1 microM) and inhibited by the transport inhibitor cum FAAH substrate AM404 with an IC(50) value of 12 microM. When a 45 s incubation time was used, the uptake of anandamide was not saturable at 37 degrees C over the concentration range tested (0.1-1 microM). Analysis of the data at 37 degrees C obtained with 45 s, 4 min and 15 min incubation times revealed a very rapid (i.e. complete by 45 s) non-saturable component followed by a slower saturable (K(m) approximately 1 microM) component of the uptake. It is concluded that the presence of a low concentration of fatty acid-free bovine serum albumin at a suitable concentration reduces non-specific binding (and release) of anandamide to cell culture wells, greatly reduces the cellular accumulation seen at 4 degrees C, and allows the visualisation of both non-saturable and saturable components of the uptake to be seen at 37 degrees C.

Arachidonoyl-serotonin inhibits in a mixed-type manner the metabolism of the endocannabinoid anandamide by the enzyme fatty acid amidohydrolase. In the present study, compounds related to arachidonoyl-serotonin have been synthesised and investigated for their ability to inhibit anandamide hydrolysis by this enzyme in rat brain homogenates. Removal of the 5-hydroxy from the serotonin head group of arachidonoyl-serotonin produced a compound (N-arachidonoyltryptamine) that was a 2.3-fold weaker inhibitor of anandamide hydrolysis, but which also produced its inhibition by a mixed-type manner (Ki(slope) 1.3 microM; Ki(intercept) 44 microM). Replacement of the amide linkage in this compound by an ester group further reduced the potency. In contrast, replacement of the arachidonoyl side chain by a linolenoyl side chain did not affect the observed potency. N-(Fur-3-ylmethyl) arachidonamide (UCM707), N-(fur-3-ylmethyl)linolenamide and N-(fur-3-ylmethyl)oleamide inhibited anandamide hydrolysis with pI50 values of 4.53, 5.36 and 5.25, respectively. The linolenamide derivative was also found to be a mixed-type inhibitor. It is concluded that the 5-hydroxy group of arachidonoyl-serotonin contributes to, but is not essential for, inhibitory potency at fatty acid amidohydrolase.

It has previously been shown that the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG) inhibit the proliferation of C6 glioma cells in a manner that can be prevented by a combination of capsazepine (Caps) and cannabinoid (CB) receptor antagonists. It is not clear whether the effect of 2-AG is due to the compound itself, due to the rearrangement to form 1-arachidonoylglycerol (1-AG) or due to a metabolite. Here, it was found that the effects of 2-AG can be mimicked with 1-AG, both in terms of its potency and sensitivity to antagonism by Caps and CB receptor antagonists. In order to determine whether the effect of Caps could be ascribed to actions upon vanilloid receptors, the effect of a more selective vanilloid receptor antagonist, SB366791 was investigated. This compound inhibited capsaicin-induced Ca2+ influx into rVR1-HEK293 cells with a pK(B) value of 6.8 +/- 0.3. The combination of SB366791 and CB receptor antagonists reduced the antiproliferative effect of 1-AG, confirming a vanilloid receptor component in its action. 1-AG, however, showed no direct effect on Ca2+ influx into rVR1-HEK293 cells indicative of an indirect effect upon vanilloid receptors. Identification of the mechanism involved was hampered by a large inter-experimental variation in the sensitivity of the cells to the antiproliferative effects of 1-AG. A variation was also seen with anandamide, which was not a solubility issue, since its water soluble phosphate ester showed the same variability. In contrast, the sensitivity to methanandamide, which was not sensitive to antagonism by the combination of Caps and CB receptor antagonists, but has similar physicochemical properties to anandamide, did not vary between experiments. This variation greatly reduces the utility of these cells as a model system for the study of the antiproliferative effects of anandamide. Nevertheless, it was possible to conclude that the antiproliferative effects of anandamide were not solely mediated by either its hydrolysis to produce arachidonic acid or its CB receptor-mediated activation of phospholipase A(2) since palmitoyltrifluoromethyl ketone did not prevent the response to anandamide. The same result was seen with the fatty acid amide hydrolase inhibitor palmitoylethylamide. Increasing intracellular arachidonic acid by administration of arachidonic acid methyl ester did not affect cell proliferation, and the modest antiproliferative effect of umbelliferyl arachidonate was not prevented by a combination of Caps and CB receptor antagonists. (C) 2003 Elsevier Inc. All rights reserved.

There is good evidence that plant-derived and synthetic cannabinoids possess neuroprotective properties. These compounds, as a result of effects upon CB(1) cannabinoid receptors, reduce the release of glutamate, and in addition reduce the influx of calcium following NMDA receptor activation. The major obstacle to the therapeutic utilization of such compounds are their psychotropic effects, which are also brought about by actions on CB(1) receptors. However, synthesis of the endogenous cannabinoids anandamide and 2-arachidonoylglycerol, which also have neuroprotective properties, are increased under conditions of severe inflammation and ischemia, raising the possibility that compounds that prevent their metabolism may be of therapeutic utility without having the drawback of producing psychotropic effects. In this review, the evidence indicating neuroprotective actions of plant-derived, synthetic and endogenous cannabinoids is presented. In addition, the pharmacological properties of endogenous anandamide-related compounds that are not active upon cannabinoid receptors, but which are also produced during conditions of severe inflammation and ischemia and may contribute to a neuroprotective action are reviewed.

The discovery of anandamide as an endogenous ligand for the cannabinoid receptors has led to a resurgence of interest in the fatty acid amides. However, N-palmitoylethanolamine (PEA), a shorter and fully saturated analogue of anandamide, has been known since the fifties. This endogenous compound is a member of the N-acylethanolamines, found in most mammalian tissues. PEA is accumulated during inflammation and has been demonstrated to have a number of anti-inflammatory effects, including beneficial effects in clinically relevant animal models of inflammatory pain. It is now engaged in phase II clinical development, and two studies regarding the treatment of chronic lumbosciatalgia and multiple sclerosis are in progress. However, its precise mechanism of action remains debated. In the present review, the biochemical and pharmacological properties of PEA are discussed, in particular with respect to its analgesic and anti-inflammatory properties.

In the present study, the effects of beta-amyloid (25-35) (Abeta (25-35)) upon calcium signalling by the human platelet has been investigated. When assays were conducted using HEPES buffers, Abeta (25-35), but not the inactive peptide Abeta (35-25), produced a robust increase in intracellular calcium that remained after removal of extracellular calcium but was abolished by the phospholipase C inhibitor U-73122. There was no significant difference between the calcium response to Abeta (25-35) in platelets from patients with Alzheimer's disease and from age-matched controls. In contrast to the robust effects on calcium mobilisation in HEPES buffers, very little calcium response to Abeta (25-35) was seen when Krebs (pH 7.8) buffer was used.

The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P3-5-phosphatase from GH3 cells, with an IC50 value of approximately 100 microM following a 10-min preincubation with the enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.

In the present study, the effects of the combination of tamoxifen ((Z)-2[p-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylamine citrate) and three cannabinoids (Delta(9)-tetrahydrocannabinol [Delta(9)-THC], cannabidiol, and anandamide [AEA]) upon the viability of C6 rat glioma cells was assessed at different incubation times and using different culturing concentrations of foetal bovine serum (FBS). Consistent with previous data for human glioblastoma cells, the tamoxifen sensitivity of the cells was increased as the FBS content of the culture medium was reduced from 10 to 0.4 and 0%. The cells expressed protein kinase C alpha and calmodulin (the concentration of which did not change significantly as the FBS concentration was reduced), but did not express estrogen receptors. Delta(9)-THC and cannabidiol, but not AEA, produced a modest reduction in cell viability after 6 days of incubation in serum-free medium, whereas no effects were seen in 10% FBS-containing medium. There was no observed synergy between the effects of tamoxifen and the cannabinoids upon cell viability.

The effect of sodium azide (NaN(3)) upon platelet Ca(2+) signalling has been investigated. A 60 s preincubation with 1 mM NaN(3) reduced the Ca(2+) response to 1 microM serotonin without a corresponding reduction in the responses to 52 mU/ml thrombin or 70 microM beta-amyloid(25-35) (A beta(25-35)). The effect of NaN(3) upon the response to serotonin, which was not blocked by either glutathione ethyl ester (GTEE) or dithiothreitol (DTT), was similar in platelets obtained from patients with Alzheimer's disease and from age- and gender-matched controls. After a preincubation time of 5 min was used, the Ca(2+) response to thrombin was greatly reduced by 1 mM NaN(3), but not by 50 microM 4-hydroxynonenal (HNE, 50 microM). Platelet levels of HNE and malondialdehyde were not significantly affected by up to 30 min of incubation with NaN(3) at room temperature. It is concluded that the rapid effect of NaN(3) upon the Ca(2+) response to serotonin in human platelets is not mediated by an inhibition of cytochrome c oxidase, and is due to an action proximal to phosphoinositide-specific phospholipase C.

beta-amyloid (A beta) peptides are known to disrupt calcium homeostasis in cells. In the present study, the effects of A beta(25-35) upon the activity of soluble Ins(1,4,5)P3-5-phosphatase have been investigated. A beta(25-35) inhibited, and dithiothreitol (DTT) increased the activity of soluble rat cerebellar Ins(1,4,5)P3-5-phosphatase. The change in activity was not accompanied by an obvious change in the sensitivity of the Ins(1,4,5)P3-5-phosphatase to inhibition by glucose-6-phosphate or phytic acid. A beta(35-25) also inhibited soluble Ins(1,4,5)P3-5-phosphatase activity, but at a lower potency than A beta(25-35). It is concluded that A beta(25-35) affects the metabolism of Ins(1,4,5)P3 although the potency is not sufficiently high to contribute to any significant extent to the effects of this peptide upon calcium homeostasis.

The original funded project (1996) had as its major goal the design and synthesis of polyammonium macrocyclic receptors for oxoanions of environmental importance, particularly those found in high level waste tanks. Nitrate was a major ion of focus. The project evolved over the last six years to target important and timely problems of critical interest to the EMSP mission. Emphasis was shifted from polyammonium receptors to lipophilic amide-based receptors more amenable for both separations and sensing applications in the last renewal, and significant effort was directed toward selective recognition of sulfate because of its detrimental role in the vitrification process. The last three years have been focused on the basic chemical aspects of anion receptor design of functional pH independent systems, with the ultimate goal of targeting the selective binding of sulfate, as well as design of separations strategies for selective and efficient removal of targeted anions. Key findings during these years include: (1) among the first synthetic sulfate-selective anion-binding agents; (2) simple, structure-based methods for modifying the intrinsic anion selectivity of a given class of anion receptors; and (3) two new classes of macrocyclic ligands, mixed amide/quaternary amine, thioamide-based macrocycles and cryptands.

In a recent study, it was found that the intrastriatal administration to rats of the organophosphorous compound soman and kainic acid produced a rapid release not only of glutamate but also of dopamine in this brain region. Dopamine is a potent source of free radicals and is known to produce cytotoxic effects, per se. This raises the possibility that the released glutamate and dopamine act synergistically to produce the neurotoxicity found after soman administration. In order to investigate the feasibility of this hypothesis in an in vitro system, the effects of dopamine and glutamate upon cell survival were investigated using chick neurons (7 DIV) in serum-free primary culture. The neurons were treated with dopamine and/or glutamate for up to 24 h and cell toxicity was then assessed either by determination of cell densities, by the release of cytoplasmic LDH or by the MTT cytotoxicity assay. L-Glutamate produced a concentration-dependent cytotoxicity that was seen as early as after 30 min of exposure, and was accompanied by an increased level of lipid peroxidation. The L-glutamate toxicity could to a large extent by prevented by NMDA receptor antagonists and to a lesser extent by catalase, superoxide dismutase or glutathione ethyl ester added 30 min before the glutamate. Dopamine was also cytotoxic, and the cytotoxicity was reduced by the combination of catalase and glutathione ethyl ester but not by the MAO inhibitors clorgyline or L-deprenyl, or by the selective dopamine uptake inhibitor GBR 12783. The cytotoxic effects of dopamine and L-glutamate were additive rather than synergistic, regardless of the incubation time used. It is concluded that chick neurons in serum-free culture are a useful in vitro model system for the study of cell toxicity produced by oxidative stress and by glutamate. The cytotoxic effects of dopamine in this model are not due to the monoamine oxidase-mediated production of hydrogen peroxide but appear at least in part to be related to oxidative stress.

The endogenous cannabimimetic anandamide is hydrolyzed by a fatty acid amide hydrolase to yield arachidonic acid and ethanolamine. In the present study, the regional distribution of the activity and its sensitivity to inhibition by the enantiomers of ibuprofen, ketorolac, and flurbiprofen has been investigated. The rate of [3H]anandamide hydrolysis was found in both 7-week-old and 90-week-old rats to be in the order hippocampuscerebral cortexcerebellumstriatum approximately midbrain, with higher rates of hydrolysis for the 7-week-old rats than for the 90-week-old rats. In whole brain (minus cerebellum), the R(-)-enantiomer of ibuprofen was a mixed-type inhibitor of anandamide hydrolysis and was approximately 2-3 times more potent than the S(+)-enantiomer, IC50 values of 230 and 750 microM, respectively, being found. A similar pattern of inhibition of anandamide hydrolysis was seen when intact C6 rat glioma cells were used. Ketorolac inhibited rat brain anandamide hydrolysis, with IC50 values of 50, 440, and 80 microM being found for the R-, S-, and R,S-forms, respectively. The IC50 value for R-flurbiprofen (60 microM) was similar to the IC50 value for the S-enantiomer (50 microM). These data demonstrate that there is no dramatic enantiomeric selectivity of NSAID compounds as inhibitors of fatty acid amide hydrolase enzyme(s) responsible for the hydrolysis of anandamide. The enantiomers of flurbiprofen and R-ketorolac are the most potent NSAID inhibitors of fatty acid amide hydrolase yet reported.

The diprotonated form of an organic-solubilized cyclo[8]pyrrole derivative, bearing eight undecyl chains on the β-pyrrolic positions, was found to extract sulfate anion effectively from neutral aqueous media into a toluene organic phase. The kinetics of sulfate anion exchange between the two phases were found to be exceedingly slow in the absence of the phase-transfer catalyst, Aliquat 336-nitrate (A336N), but appreciable in its presence. The bisnitrate anion bound form of this cyclo[8]pyrrole could be generated in situ by subjecting the toluene phase containing initially 0.5 mM of the sulfate anion bound form and 0.1 mM trioctylamine (TOA) to successive equilibrations with aqueous 0.1 M HNO3 until sulfate was no longer detected in the aqueous phase. This bisnitrate complex, when studied as a 0.5 mM solution in toluene in the presence of 0.1 mM (TOAH)+(NO3-), was also found to be an effective extractant for sulfate anion. DSO4 values of 0.001 and 1000 were observed at 1 M NaNO3(aq) and 0.3 mM NaNO3(aq), r...

Relatively little is known about the properties of the NMDA receptor in avian species. In the present study, NMDA receptor pharmacology and function has been studied in the embryonic chick brain. The competitive antagonist ligand [3H]CGP 39653 bound to telencephali membranes from 17-day-old chick embryos with KD and Bmax values of 6.6 nM and 3.3 pmol/mg protein, respectively. The binding was inhibited by CGS 19755, L-glutamate, R-CPP and D-AP5 with pI50 values of 7.57, 7.49, 7.28 and 7.08, respectively. L-glycine, S-AMPA, kainate and MK-801 only weakly affected the binding. The inhibitory potency of NMDA (pI50 value 6.98), however, was greater than seen in other species, suggesting a species difference in receptor structure. Such a possibility was investigated by use of two NMDA-R1 antibodies, AB 1516 and 69921 A, which recognize the C-terminal region and an intracellular domain of the rat receptor, respectively. Immunoreactive signals (approximately 116 kDa) were found for a rat brain homogenate, whereas only 69921A gave an immunoreactive signal in the chick. The functional status of the NMDA receptors was investigated in serum-free cultures of neurons from 8-day-old chick embryos. Glutamate and NMDA were found concentration-dependently, and in a manner that could be antagonized by CGS 19755 and MK-801, to produce cell death. Thus, the present study indicates that chick embryonic brain expresses functional NMDA receptors that are pharmacologically and possibly structurally different from their rodent counterparts.

Great advances have been made in recent years in our knowledge of the genetic mutations found in early onset familial Alzheimer's disease (AD) and their pathological consequences. The pathogenesis of sporadic AD, on the other hand, is less clear, although a central role of oxidative stress is indicated. In the AD brain, severe dysfunctions in the phosphoinositide signalling pathway have been reported. In view of the fact that (a) oxidative stress can adversely affect phosphoinositide breakdown and hence diacylglycerol-mediated activation of protein kinase C and (b) protein kinase C activation reduces the production of beta-amyloid peptide from amyloid precursor protein, it is possible that this represents a pathogenic pathway whereby oxidative stress can lead to amyloid deposition and the development of the disease.

The intracellular calcium indicator dye Fura-2 has been widely used for the study of human platelet thrombin receptor-mediated calcium mobilisation in disease states. In general, authors (a) use a Fura-2/AM concentration of 2–3 μmol/l and (b) calibrate fluorescence signals on the basis of maximum and minimum ratios of fluorescence (Rmax and Rmin) and the ratio of the calcium-free and calcium-bound fluorescence at an excitation wavelength of 380 nm ( “C 2 /B 2 ” ). In the present study, it is found (a) that a greater peak response to thrombin is seen when 1 μmol/l Fura-2/AM rather than 2 or 3 μmol/l is used; and (b) that calibration leads to a poorer test-retest reliability and in general a greater variability of the obtained calcium signal than when the simple measurement of the 340 nm/380 nm fluorescence ratio is used. It is suggested that this poor variability is due to the presence of an extracellular factor that can quench the Fura-2 signal once the platelets have been permeabilised by detergent treatment. Consistent with this, addition of bovine serum albumin to the assay medium has no significant effect on the fluorescence ratio response to thrombin, but greatly increases the observed calibrated calcium signal.

Recent research into the pathogenesis of Alzheimer's disease (AD) has focused upon the proposed neurotoxic role played by the beta-amyloid peptide. Other important neurochemical disturbances, such as the dramatic breakdown of intracellular signalling, should however not be overlooked. In the present article, the authors attempt to investigate the relationship between signalling disturbances and beta-amyloid and ask the question 'which came first?' The available evidence is discussed, and it is concluded that the need for a wide, rather than restricted, focus on biochemical events associated with AD is required if progress is to be made in the understanding and eventual treatment of this disease.

It has long been assumed that widespread changes in postsynaptic neurotransmitter receptor function are not a feature of the disrupted neurotransmission seen in the brains with Alzheimer's disease (AD). However, recent evidence from postmortem brain and fibroblast studies suggests that both the neurotransmitter receptor/G-protein-modulated adenylyl cyclase and the phosphatidylinositol hydrolysis signal transduction cascades are disrupted in AD. Such disruptions may severely limit the functional integrity of key receptor types and undermine pharmacological attempts to ameliorate disease symptomatology through neurotransmitter replacement strategies. The involvement of some signalling mechanisms in the regulation of beta-amyloid precursor protein metabolism suggests also that disrupted signal transduction may exacerbate AD pathology.

The fatty acid amide class of compounds, which include the endocannabinoid anandamide and the sleep-inducing compound oleamide, have been shown in vitro to have a multiplicity of actions upon different neurochemical systems. In the present issue of this journal, Leggett et al present data indicating that oleamide functionally activates CB(1) cannabinoid receptors in vitro. The significance of their finding is discussed in this commentary.

The blockade of veratrine-stimulated phosphoinositide breakdown in rat cerebral cortical miniprisms as a model of local anesthetic actions on voltage-dependent sodium channels was assessed. Veratrine stimulated phosphoinositide breakdown with an EC50 value of 5 micro Meter. The stimulation produced by 20 micro Meter veratrine was blocked completely by (+)-bupivacaine (IC50 7.6 micro Meter [mean of three separate experimental series]), (-)-bupivacaine (IC50 7.3 micro Meter), lidocaine (IC50 34 micro Meter), etidocaine (IC50 3.4 micro Meter), tetracaine (IC50 approximate 2 micro Meter), and prilocaine (IC50 110 micro Meter). Phosphoinositide breakdown responses to ouabain (100-1000 micro Meter) and K+ (50 mM) were only partially blocked by (+)-bupivacaine, and the responses to monensin (100 and 1000 micro Meter) and noradrenaline (30 micro Meter) were not blocked at all by this drug. Nifedipine produced no significant effects on the phosphoinositide response to 10 micro Meter veratrine. It is concluded that in pulse label experiments using rat cerebral cortical miniprisms, local anesthetics in general, and (+)-bupivacaine in particular, block the phosphoinositide response to veratrine with a high degree of specificity. This system may be useful as a relatively simple and quantitative assay for drug effects on Na+-channels. (Anesth Analg 1995;81:480-5)

Levels of the calcium mobilising receptors for the phosphoinositide hydrolysis derived second messengers, inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and inositol(1,3,4,5) tetrakis-phosphate [Ins(1,3,4,5)P4] were compared in the cerebellum, superior temporal and superior frontal cortex of a series of Alzheimer's disease and matched control cases. Membrane [3H]Ins(1,4,5)P3 radioligand binding experiments performed under steady state conditions revealed that the number of Ins(1,4,5)P3 recognition sites was significantly decreased in all three brain regions of the Alzheimer's disease cases, compared to controls. In contrast, [3H]Ins(1,3,4,5)P4 binding levels, as assessed in competition analyses, were not significantly different between the groups in any brain region. Moreover, the Hill coefficients for inhibition of [3H]Ins(1,3,4,5)P4 binding by non-radioactive Ins(1,3,4,5)P4 were less than unity in both the control and Alzheimer's disease brains, suggesting that the heterogeneity of these binding sites are also maintained in the disease. It is concluded that disruptions of the phosphoinositide hydrolysis pathway in Alzheimer's disease brain are associated with a selective loss of calcium mobilising Ins(1,4,5)P3, but not Ins(1,3,4,5)P4 receptor sites. These alterations may contribute to an altered calcium homeostasis in Alzheimer's disease, as well as providing one reason for the lack of success of cholinergic replacement therapies aimed at enhancing muscarinic receptor-mediated phosphatidylinositol hydrolysis.

Local anaesthetics and opioid drugs function synergistically to provide analgesia. In the present study, the nature of this synergy has been investigated using in vitro radioligand binding to determine whether the local anaesthetics bupivacaine and tetracaine modulate the binding of two kappa-opioid receptor ligands, [3H]U-69593 (5-alpha,7-alpha,8-beta-(-)-N-methyl-N[7-(1-pyrrolodinyl)-1-oxa spiro(4,5)dec-8-yl]-benzene acetamide) and [3H](-)-EKC (ethylketocyclazocine). [3H]U-69593 bound with a KD of 0.88 nM and a Bmax of 2.39 +/- 0.22 fmol/mg wet weight in guinea pig cerebellar membranes. The binding was inhibited by bremazocine and morphine with Hill slopes near unity and pI50 values of 9.96 and 6.84-6.86, respectively. [3H]U-69593 binding was inhibited by Gpp(NH)p (5'-guanylyl imidodiphosphate) and NaCl, consistent with an agonist action of the compound. The binding characteristics of the ligand were not changed by bupivacaine or tetracaine. [3H](-)-EKC bound with KD values of 0.55 and 0.97 nM and Bmax values of 4.22 and 0.99 fmol/mg wet weight in guinea pig cerebellar membranes and rat spinal cords, respectively. In the rat spinal cord, [3H](-)-EKC appeared to act as an agonist/antagonist, since the presence of Gpp(NH)p and NaCl only produced a small (21%) reduction in binding, but reduced the pI50 for the residual binding to inhibition by morphine from 6.33-6.39 to 5.95. As with [3H]U-69593, the binding characteristics of [3H](-)-EKC were not affected by bupivacaine or tetracaine. These studies demonstrate that effects of kappa-opioid receptor recognition site conformation are unlikely to explain the clinically observed synergy between local anaesthetics and opioids.

The characteristics of specific [3H]Ins(1,3,4,5)P4 binding sites in human cerebellar membranes were determined in this study. Binding rapidly reached steady state, possessed a pH optimum of 4.5-5.1 and was greater in the absence of BSA than in its presence. Heparin inhibited both specific and pseudospecific binding of the ligand, whereas only the specific binding was inhibited by non-radioactive Ins(1,3,4,5)P4. Calcium at a concentration of 1 mM, reduced binding by 27%. Competition studies with other inositol phosphates showed specificity for Ins(1,3,4,5)P4 with a pI50 value of 6.87 and a Hill coefficient of 0.27, indicating two sites. Ins(1,2,5,6)P4, Ins(1,3,4,6)P5, Ins(3,4,5,6)P4 displaced binding with IC50 values ranging from 0.1-1 microM, Ins(1,2,5,6)P4 and Ins(1,3,4,5,6)P5 being the most potent. Ins(1,4)P2 and Ins(1,5,6)P3 had lesser effects on binding. Rosenthal analysis of [3H]Ins(1,3,4,5)P4 saturation binding data at low ligand concentrations gave a KD of 27 nM and a Bmax of 33 pmol/mg protein. It is concluded that [3H]Ins(1,3,4,5)P4 binding sites in human cerebellar membranes have similar characteristics to these sites reported in the literature in animal cerebellar tissue, but are in greater abundance.

In the present study the relative densities of a number of G protein subunits were quantified in membranes prepared from the hippocampus, temporal cortex and angular gyrus of Alzheimer's disease and control post-mortem brain by immunoblotting with specific polyclonal antisera against Gsα, Giα, Giα-1, Goα and Gs protein subunits. In addition, basal, Gs-stimulated and Gi-inhibited adenylyl cyclase activities were measured in the same hippocampal membrane samples. Densitometric analysis of the immunoblot data revealed a 58% reduction in the levels of Giα, and a 75% reduction in the levels of Giα-1, in the Alzheimer's disease temporal cortex. Giα levels were reduced, by 37% in the angular gyrus of the Alzheimer's disease cases. The ratio of large to small molecular weight isoforms of the Gsα subunit was significantly increased in both the hippocampus and the angular gyrus of the Alzheimer's disease samples when compared to control values, although the difference in individual Gsα isoform levels did not attain statistical significance when comparing groups. No statistically significant differences were observed in Goα or Gs levels when comparing control and Alzheimer's disease cases. Gs-stimulated adenylyl cyclase activity was significantly reduced in the Alzheimer's disease samples compared to controls, whereas Gi-inhibited adenylyl cyclase activity was unchanged. No significant differences were observed between the control and Alzheimer's disease samples for either basal or forskolin stimulated adenylyl cyclase activity. The ratio of hippocampal Gs-stimulated to basal adenylyl cyclase activity correlated significantly with the large to small Gsα subunit ratio. We conclude that G protein subunit levels are selectively altered in different regions of the Alzheimer's disease brain. These alterations may contribute to the functional changes observed in the CNS in Alzheimer's disease by modifying a number of signal transduction pathways.

Following a preincubation period of 10 min, disulfiram and its analogues FLA 46, FLA 63, FLA 99, EWP 815 and EWP 840 inhibited the breakdown of 10 microM [3H]Ins(1,4,5)P3 by Ins(1,4,5)P3 5-phosphatase from GH3 cells, with IC50 values (in microM), for soluble/particulate enzymes respectively, of: disulfiram, 24/24; FLA 46, 23/30; FLA 63, 24/6; FLA 99, 50/48; EWP 815, 8/6; EWP 840, 11/8. The inhibition produced by FLA 99 was time-dependent in nature, although inhibition was found in the absence of a preincubation period. EWP 815 and EWP 840 were more potent inhibitors of Ins(1,4)P2 phosphatase than of Ins(1,4,5)P3 5-phosphatase. Thyrotropin-releasing hormone (TRH; 3/100 microM)-stimulated inositol phospholipid breakdown in prelabelled GH3 cells was inhibited by disulfiram (IC50 values 63/52 microM respectively), FLA 46 (89/110 microM), EWP 815 (83/71 microM) and EWP 840 (220/200 microM), without affecting basal breakdown rates. FLA 99 did not inhibit either basal or TRH-stimulated activity at any of the concentrations tested (30, 100 and 300 microM). [3H]Ins(1,4,5)P3 binding to its cerebellar receptor was not inhibited by any of the compounds over a concentration range of 3-300 microM, although an increased level of binding was seen at high concentrations. FLA 99 and EWP 840 increased the basal intracellular Ca2+ concentration in GH3 cells, but with no corresponding effect on the Ca2+ response to TRH stimulation. These compounds did not increase the cellular permeability to Trypan Blue, but did affect cell proliferation. It is concluded that disulfiram and related compounds produce dramatic effects on Ins(1,4,5)P3 metabolism in GH3 cells.

The effects of agonal status, postmortem delay, and age on human brain adenylyl cyclase activity were determined in membrane preparations of frontal cortex from a series of 18 nondemented subjects who had died with no history of neurological or psychiatric disease. Basal and guanosine 5′-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were not significantly reduced over an interval from death to postmortem of between 3 and 37 h and were also not significantly different between individuals dying with a long terminal phase of an illness and those dying suddenly. Basal and aluminum fluoride-stimulated enzyme activities showed a negative correlation with increasing age of the individual. In subsequent experiments, basal and guanosine 5′-O-(3-thiotriphosphate)-, aluminum fluoride-, and forskolin-stimulated enzyme activities were compared in five brain regions from a series of eight Alzheimer's disease and seven matched nondemented control subjects. No significant differences were observed between the groups for either basal activity or activities in response to forskolin stimulation of the catalytic subunit of the enzyme. In contrast, enzyme activities in response to stimulation with guanosine 5′-O-(3-thiotriphosphate) and aluminum fluoride were significantly reduced in preparations of neocortex and cerebellum from the Alzheimer's disease cases compared with the nondemented controls. Lower guanosine 5′-O-(3-thiotriphosphate)-, but not aluminum fluoride-, stimulated activity was also observed in preparations of frontal cortex from a group of four disease controls compared with nondemented control values. The disease control group, which contained Parkinson's disease and progressive supranuclear palsy patients, showed increased forskolin-stimulated activity compared with both the nondemented control and the Alzheimer's disease groups. These findings indicate a widespread impairment of G protein-stimulated adenylyl cyclase activity in Alzheimer's disease brain, which occurs in the absence of altered enzyme catalytic activity and which is unlikely to be the result of non-disease-related factors associated with the nature of terminal illness of individuals.

In recent years, there have been a large number of clinical trials aimed at alleviating the symptoms of dementia of the Alzheimer type (DAT), a disorder affecting more than 5% of the population over the age of 65 years. Despite a wide variety of treatment strategies, ranging from neurotransmitter (preferentially cholinergic) replacement therapy through hormonal treatments to metabolic activators (see Table 1 for examples), results of clinical trials have generally been disappointing (for reviews, see Bagne et al., 1986; Gottfries, 1989; Whalley, 1989). Such poor outcomes of treatments raise the question as to whether dementia of the Alzheimer type is a treatable condition. In the present chapter, this question has been examined with respect to the neurotransmitter replacement strategies.

The coupling of alpha 2-adrenoceptors to guanine nucleotide binding G-proteins was investigated in cerebral cortical membranes from control and Alzheimer's disease brain by characterizing the effects of MnCl2 and Gpp[NH]p on [3H]clonidine binding. The manganese induced elevation of [3H]clonidine binding was apparent in both control and Alzheimer's disease samples and the effect showed no significant difference between the two groups in the frontal cortex. However, the MnCl2 concentration dependence curves for control and Alzheimer's disease samples were significantly different from one another in the temporal cortex, whereas the pattern of stimulation by MnCl2 remained the same. The guanine nucleotide analogue, Gpp[NH]p inhibited [3H]clonidine binding in a concentration-dependent manner, the profiles of inhibition showing no significant differences between control and Alzheimer's disease samples. Analysis of the effect of Gpp[NH]p on [3H]clonidine saturation binding curves showed no significant differences between control and Alzheimer's disease samples in either frontal (Kd = 9.68 +/- 1.38, 9.1 +/- 2.6 nM; Bmax = 40.23 +/- 4.33, 44.3 +/- 9.4 fmol/mg, control and Alzheimer's disease values, respectively), or temporal (Kd = 11.61 +/- 4.04, 5.38 +/- 2.5 nM; Bmax = 52.0 +/- 14.0, 31.07 +/- 8.00 fmol/mg control and Alzheimer's disease values, respectively) cortices.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously reported a reduction in the inhibitory effect of somatostatin on adenylyl cyclase activity in the superior temporal cortex of a group of Alzheimer's disease cases, compared to a group of matched controls. In the present study, the levels of high affinity 125I-Tyr11-somatostatin-14 binding, its modulation by guanine nucleotides and the effects of somatostatin on adenylyl cyclase activity have been measured in preparations of frontal cortex, hippocampus, caudate nucleus and cerebellum from the same patient and control groups. A significant reduction in 125I-Tyr11-somatostatin-14 binding was observed in the frontal cortex, but not other regions, of the Alzheimer's disease group, compared with control values. The profiles of inhibition of specific 125I-Tyr11-somatostatin-14 binding by Gpp(NH)p were similar in all regions in both groups. No significant differences in basal, forskolin-stimulated, or somatostatin and neuropeptide Y inhibitions of adenylyl cyclase activity were found between the two groups. The pattern of change of somatostatin binding in the Alzheimer's disease cases observed in the present study differs from the reported pattern of loss of somatostatin neurons and may be secondary to the degeneration of somatostatin receptor-bearing cholinergic afferents arising from the nucleus basalis. The results of this study indicate that impaired somatostatin modulation of adenylyl cyclase is not a global phenomenon in Alzheimer's disease brain and also that there are no major disruptions of somatostatin receptor-G-protein coupling or of adenylyl cyclase catalytic activity in this disorder.

The activity of a phospholipase C which hydrolyses exogenous phosphatidylinositol-4,5-bisphosphate [( 3H]PtdIns(4,5)P2) in membranes prepared from frozen postmortem human brain and rat brain was investigated. Enzyme characteristics were essentially similar in membranes prepared from frozen postmortem brain and fresh or frozen rat brain. The [3H]PtdIns(4,5)P2 solubilization and assay procedure employed resulted in an efficient availability of the substrate for the enzyme. The non-hydrolysable guanosine triphosphate analogue guanosine 5'-[beta gamma-imido]diphosphate (Gpp[NH]p) stimulated hydrolysis rapidly with a half maximum activity of approximately 25 microM. This stimulation was not specific for guanine nucleotides as ATP, imidodiphosphate and pyrophosphate also caused enzyme activation. However these activation effects could be distinguished by the polyanion spermine. The non-hydrolysable guanine dinucleotide analogue guanosine 5'-[beta-thio]diphosphate acted as a partial agonist thereby inhibiting the stimulatory effect of Gpp[NH]p. Gpp[NH]p-stimulated enzyme activity showed a maximum response in the presence of 1 mM deoxycholate and displayed a pH optima in the range 7.0-7.5. PtdIns(4,5)P2 hydrolysis was observed in the absence of added calcium, but hydrolytic cleavage was inhibited in the presence of divalent ion chelators. Magnesium inhibited PtdIns(4,5)P2 hydrolysis in a concentration-dependent manner. Elucidation of these aspects of the phosphatidylinositol cycle in normal human postmortem brain will permit comparative studies in CNS disease states.