Potent beta-amyloid modulators.

Currently, the potential to interfere with the pathology of beta-amyloid targeting a well-known drugable enzyme, the acetylcholinesterase (AChE), is opened. Peripheral or dual binding site inhibitors of AChE may simultaneously alleviate the cognitive and behavioral deficits in Alzheimer's disease (AD) patients and, more importantly, act as disease-modifying agents delaying amyloid plaque formation. As part of a rational drug design program directed to find dual binding site AChE inhibitors, several families of compounds have been synthesized as potent AChE inhibitors. From these series, several drug candidates were selected based on their potent and selective inhibition of AChE (subnanomolar activity) and their interference with the beta-amyloid aggregation in vitro (IC(50) in the low micromolar range). First in vivo data confirm our initial hypothesis. Oral treatment with NP-61 for 3 months is able to reverse the cognitive impairment (Morris water maze test) and to reduce plaque load in the brains of human amyloid precursor protein transgenic mice (Swedish mutation). These results suggest that NP-61, a potent beta-amyloid modulator, is able to reverse the AD-like neurodegenerative phenotype in transgenic mice, indicating a promising disease-modifying agent for clinical application.

Donepezil-tacrine hybrid related derivatives as new dual binding site inhibitors of AChE.

A new series of donepezil-tacrine hybrid related derivatives have been synthesised as dual acetylcholinesterase inhibitors that could bind simultaneously to the peripheral and catalytic sites of the enzyme. These new hybrids combined a tacrine, 6-chlorotacrine or acridine unit as catalytic binding site and indanone (the heterocycle present in donepezil) or phthalimide moiety as peripheral binding site of the enzyme, connected through a different linker tether length. One of the synthesised compounds emerged as a potent and selective AChE inhibitor, which is able to displace propidium in a competition assay. These results seem to confirm the ability of this inhibitor to bind simultaneously to both sites of the enzyme and make it a promising lead for developing disease-modifying drugs for the future treatment of Alzheimer's disease. To gain insight into the molecular determinants that modulate the inhibitory activity of these compounds, a molecular modelling study was performed to explore their binding to the enzyme.

Differential expression of calcium channel subtypes in the bovine adrenal medulla.

This study aimed at determining the distribution and expression levels of different subtypes of Ca(2+) channels in the bovine adrenal medulla, and whether individual subtypes were more abundant in chromaffin cells exhibiting an adrenergic or a noradrenergic phenotype. In situ hybridization using riboprobes specific for the pore-forming Ca(2+) channel alpha(1D) (L-type channel), alpha(1B) (N-type channel), and alpha(1A) (P/Q-type channel) subunits of bovine chromaffin cells showed a broad distribution of the three transcripts in adrenal medulla tissue. However, a tissue-specific expression pattern of individual subunits was found; whereas alpha(1B) mRNA was homogeneously distributed throughout the medulla, alpha(1D) and alpha(1A) transcripts were present at higher densities in the internal medullary area, far away from the adrenal cortex. These results were corroborated by comparative analysis of the alpha(1B), alpha(1D), and alpha(1A) products amplified by RT-PCR from total RNA extracted from small pieces of tissue dissected out from external or internal medullary areas. Interestingly, immunohistochemical experiments performed in adrenal gland sections, using antidopamine-beta-hydroxylase and anti-phenylethanolamine-N-methyltransferase antibodies, indicated a higher density of noradrenergic over adrenergic chromaffin cells in the internal medullary region. These results provide direct evidence in favor of a heterogeneous distribution of Ca(2+) channel subtypes in the adrenal medulla, in agreement with previous functional data showing that blockade of the high K+ -elicited responses by dihydropyridines was greater in noradrenergic than in adrenergic chromaffin cells. These differences may be relevant for the differential release regulation of each catecholamine under physiological and pathophysiological conditions.

Greater diversity than previously thought of chromaffin cell Ca2+ channels, derived from mRNA identification studies.

Using reverse transcription followed by PCR amplification (RT-PCR), we have identified multiple messenger RNAs encoding for the neuronal pore-forming Ca(2+) channel subunits alpha(1A) (P/Q channel), alpha(1B) (N channel), alpha(1D) (neuronal/endocrine L channel), alpha(1E) (R channel), alpha(1G-H) (T channel) and alpha(1S) (skeletal muscle L channel) in bovine chromaffin cells. mRNAs for the auxiliary beta(2), beta(3), beta(4), alpha(2)/delta and gamma(2) subunits were also identified. In agreement with these molecular data, perforated patch-clamp recordings of whole-cell Ca(2+) currents reveal the existence of functional R-type Ca(2+) channels in these cells that were previously undetected with other techniques. Our results provide a molecular frame for a much wider functional diversity of Ca(2+) channels in chromaffin cells than that previously established using pharmacological and electrophysiological approaches.

Predictive value of nuclear factor kappaB activity and plasma cytokine levels in patients with sepsis.

The relationship between fluctuating cytokine concentrations in plasma and the outcome of sepsis is complex. We postulated that early measurement of the activation of nuclear factor kappaB (NF-kappaB), a transcriptional regulatory protein involved in proinflammatory cytokine expression, may help to predict the outcome of sepsis. We determined NF-kappaB activation in peripheral blood mononuclear cells of 34 patients with severe sepsis (23 survivors and 11 nonsurvivors) and serial concentrations of inflammatory cytokines (interleukin-6, interleukin-1, and tumor necrosis factor) and various endogenous antagonists in plasma. NF-kappaB activity was significantly higher in nonsurvivors and correlated strongly with the severity of illness (APACHE II score), although neither was related to the cytokine levels. Apart from NF-kappaB activity, the interleukin-1 receptor antagonist was the only cytokine tested whose level in plasma was of value in predicting mortality by logistic regression analysis. These results underscore the prognostic value of early measurement of NF-kappaB activity in patients with severe sepsis.

Multiple calcium pathways induce the expression of SNAP-25 protein in chromaffin cells.

Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24-48 h enhanced 2.5 to five times the expression of SNAP-25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L-type Ca2+ channel blocker) but was unaffected by either omega-conotoxin GVIA (an N-type Ca2+ channel blocker) or -agatoxin IVA (a P/Q-type Ca2+ channel blocker). Combined blockade of N and P/Q channels with omega-conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of fumidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP-25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L-type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo-1-loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.

Differential blockade of rat alpha3beta4 and alpha7 neuronal nicotinic receptors by omega-conotoxin MVIIC, omega-conotoxin GVIA and diltiazem.

Rat alpha3beta4 or alpha7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non-toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 microM) applied at regular intervals. The N/P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited alpha3beta4 currents with an IC50 of 1.3 microM; the blockade was non-competitive and reversible. The alpha7 currents were unaffected. At 1 microM, omega-conotoxin GVIA (N-type Ca2+ channel blocker) inhibited by 24 and 20% alpha3beta4 and alpha7 currents, respectively. At 1 microM, omega-agatoxin IVA (a P/Q-type Ca2+ channel blocker) did not affect alpha7 currents and inhibited alpha3beta4 currents by only 15%. L-type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on alpha3beta4 nicotinic AChRs. The mechanism of alpha3beta4 currents blockade by omega-conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage-dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while omega-toxins did not. These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.

Unmasking the functions of the chromaffin cell alpha7 nicotinic receptor by using short pulses of acetylcholine and selective blockers.

Methyllycaconitine (MLA), alpha-conotoxin ImI, and alpha-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 microM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC50s of 100 nM for MLA and 300 nM for alpha-conotoxin ImI and alpha-bungarotoxin. MLA (100 nM), alpha-conotoxin ImI (1 microM), and alpha-bungarotoxin (1 microM) halved the entry of 45Ca2+ stimulated by 5-s pulses of 300 microM ACh applied to incubated cells. These supramaximal concentrations of alpha7 nicotinic receptor blockers depressed by 30% (MLA), 25% (alpha-bungarotoxin), and 50% (alpha-conotoxin ImI) the inward current generated by 1-s pulses of 100 microM ACh, applied to voltage-clamped chromaffin cells. In Xenopus oocytes expressing rat brain alpha7 neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, alpha-conotoxin ImI, and alpha-bungarotoxin with IC50s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through alpha3 beta4 nAChR was unaffected by alpha-conotoxin ImI and alpha-bungarotoxin, and weakly blocked by MLA (IC50 = 1 microM). The functions of controlling the electrical activity, the entry of Ca2+, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to alpha3 beta4 nAChR; the present results constitute the first evidence to support a prominent role of alpha7 nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.

Capacitative Ca2+ entry into Xenopus oocytes is sensitive to omega-conotoxins GVIA, MVIIA and MVIIC.

We have studied capacitative Ca2+ entry into Xenopus oocytes by depleting intracellular Ca2+ stores with inositol 1,4,5-trisphosphate or thapsigargin. Capacitative Ca2+ entry was evoked by hyperpolarisation and monitored via the Ca(2+)-activated Cl- current. Hyperpolarisation-evoked currents increased with extracellular [Ca2+] in the range 0.9-5 mM, and were reversibly inhibited by extracellular Mg2+ (0.1-10 mM) by up to 60%. Currents were decreased by the voltage-gated Ca2+ channel antagonists omega-conotoxin GVIA, MVIIA and MVIIC (0.3-10 microM) and the inhibition of Ca2+ entry in individual oocytes by omega-conotoxins GVIA and MVIIA was highly heterogeneous, but not additive. Flunarizine (10 microM) and the imidazoles SK&F 96365 (10 microM), miconazole (40 microM) and econazole (40 microM) partly blocked Ca2+ entry. Ca2+ entry was unaffected by calciseptine (300 nM) or alpha-bungarotoxin (1 microM). The possibility that these compounds might inhibit the Ca(2+)-activated Cl- current rather than capacitative Ca2+ entry itself was examined by recording the Cl- current activated by the increase in [Ca2+]i activated by the flash photolysis of caged Ca2+. Eicosatetraynoic acid (2-10 microM) markedly inhibited, and La3+ (1 mM but not 100 microM) potentiated the increase in Ca(2+)-activated Cl- current. In contrast, omega-conotoxins and Mg2+ had no effect on the Ca(2+)-activated Cl- current itself. These findings support the hypothesis that capacitative Ca2+ entry into Xenopus oocytes occurs through channels with a pharmacology similar to that of neuronal non-L type voltage-gated Ca2+ channels.

Serotonergic effects of dotarizine in coronary artery and in oocytes expressing 5-HT2 receptors.

In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 microM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 microM, respectively. Flunarizine inhibited both types of contractions with IC50 values of 1.7 microM for 5-HT and 2.4 microM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2(+)-activated Cl- channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 microM dotarizine and blocked around 62% by 10 microM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 microM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.

Atypical location of cannabinoid receptors in white matter areas during rat brain development.

Previous evidence suggests that the endogenous cannabinoid system could emerge and be operative early during brain development. In the present study, we have explored the distribution of specific binding for cannabinoid receptors in rat brain at gestational day 21 (GD21), postnatal days 5 (PND5) and 30 (PND30), and at adult age (> 70 days after birth) by using autoradiography with [3H]CP-55,940. Our results indicated that specific binding for cannabinoid receptors can be detected in the brain of rat fetuses at GD21 in the classic areas that contain these receptors in adulthood-in particular, in the cerebellum and the hippocampus and, to a lesser extent, in the basal ganglia, several limbic structures, and cerebral cortex. The density of cannabinoid receptors in all these structures increased progressively at all postnatal ages studied until reaching the classical adult values in 70-day-old animals. Interestingly, cannabinoid receptor binding can also be detected at GD21 in regions, in which they are scarcely distributed or not located in the adult brain and that have the particularity of all being enriched in neuronal fibers. Among these were the corpus callosum, anterior commissure, stria terminalis, fornix, white matter areas of brainstem, and others. This atypical location was quantitatively high at GD21, tended to wane at PND5, and practically disappeared at PND30 and in adulthood, with the only exception being the anterior commissure, which exhibited a moderate density for cannabinoid receptors. Moreover, the binding of [3H]CP-55,940 to cannabinoid receptors in the white matter regions at GD21 seems to be functional and involves a GTP-binding protein-mediated mechanism. Thus, the activation of these receptors with an agonist such as WIN-55,212-2 increased the binding of [35S]-guanylyl-5'-O-(gamma-thio)-triphosphate, measured by autoradiography, in the corpus callosum and white matter areas of brainstem of fetuses at GD21. This increase was reversed by coincubation of WIN-55,212-2 with SR141716, a cannabinoid receptor antagonist. As this antagonist is specific for the cerebral cannabinoid receptor subtype, called CB1, we can assert that the signal found for cannabinoid receptor binding in the fetal and early postnatal brain likely corresponds to this receptor subtype. Collectively, all these data suggest the existence of a transient period of the brain development in the rat, around the last days of the fetal period and the first days of postnatal life, in which CB1 receptors appear located in neuronal fiber-enriched areas. During this period, CB1 receptors would be already functional acting through a GTP-binding protein-mediated mechanism. After this transient period, they progressively acquire the pattern of adult distribution. All this accounts for a specific role of the endogenous cannabinoid system in brain development.

Effects of chronic exposure to delta9-tetrahydrocannabinol on cannabinoid receptor binding and mRNA levels in several rat brain regions.

Previous data showed the development of tolerance to a variety of pharmacological effects of plant and synthetic cannabinoids when administered chronically. This tolerance phenomenon has been related both to enhancement of cannabinoid metabolism and, in particular, to down-regulation of brain CB1 cannabinoid receptors, although this has been only demonstrated in extrapyramidal areas. In the present study, we have tested, by using autoradiographic analysis of CB1 receptor binding combined with analysis of CB1 receptor mRNA levels in specific brain regions by Northern blot, whether the reduction in binding levels of CB1 receptors observed in extrapyramidal areas after a chronic exposure to delta9-tetrahydrocannabinol (delta9-THC), also occurs in most brain areas that contain these receptors. Results were as follows. The acute exposure to delta9-THC usually resulted in no changes in the specific binding of CB1 receptors in all brain areas studied, discarding a possible interference in binding kinetic of the pre-bound administered drug. The only exceptions were the substantia nigra pars reticulata and the cerebral cortex, which exhibited decreased specific binding after the acute treatment with delta9-THC presumably due to an effect of the pre-bound drug. The specific binding measured in animals chronically (5 days) exposed to delta9-THC decreased ranging from approximately 20 up to 60% of the specific binding measured in control animals in all brain areas. Areas studied included cerebellum (molecular layer), hippocampus (CA1, CA2, CA3, CA4 and dentate gyrus), basal ganglia (medial and lateral caudate-putamen and substantia nigra pars reticulata), limbic nuclei (nucleus accumbens, septum nucleus and basolateral amygdaloid nucleus), superficial (CxI) and deep (CxVI) layers of the cerebral cortex and others. There were only two brain regions, the globus pallidus and the entopeduncular nucleus, where the specific binding for CB receptors was unaltered after 5 days of a daily delta9-THC administration. In addition, we have analyzed the levels of CB1 receptor mRNA in specific brain regions of animals chronically exposed to delta9-THC, in order to correlate them with changes in CB1 receptor binding. Thus, we observed a significant increase in CB1 receptor mRNA levels, but only in the striatum, with no changes in the hippocampus and cerebellum. In summary, CB1 receptor binding decreases after chronic delta9-THC exposure in most of the brain regions studied, although this was not accompanied by parallel decreases in CB receptor mRNA levels. This might indicate that the primary action of delta9-THC would be on the receptor protein itself rather than on the expression of CB1 receptor gene. In this context, the increase observed in mRNA amounts for this receptor in the striatum should be interpreted as a presumably compensatory effect to the reduction in binding levels observed in striatal outflow nuclei.

Involvement of GABA(B) receptors in the motor inhibition produced by agonists of brain cannabinoid receptors.

The present study was designed to investigate the possibility of activation of GABA(B) receptors during the motor inhibition caused by cannabimimetics. Adult male rats were injected with an acute dose of arachidonylethanolamide (AEA), Delta(9)- tetrahydrocannabinol (THC), beclofen or vehicle, after pretreatment with CGP 35348, a specific antagonist for GABA(B) receptors, or vehicle, and the behavioral response produced by these compounds tested in an open field. As expected, the administration of either AEA or THC produced a very pronounced motor inhibition, reflected by decreased ambulation and increased time spent in inactivity. The administration of baclofen also produced a marked motor deficit, with similar changes to those observed with both cannabimimetics. Pretreatment with the GABA(B) antagonist, CGP 35348, prevented the motor inhibition induced by baclofen and also attenuated the motor deficit caused by both cannabimimetics, suggesting a role for this receptor. In summary, a GABAergic influence, acting through GABA(B) receptors, seems to be involved in mediating motor effects of cannabimimetics, since the blockade of these receptors attenuates cannabimimetic-induced signs of motor inhibition.

Extrapyramidal effects of methanandamide, an analog of anandamide, the endogenous CB1 receptor ligand.

Recent evidence has demonstrated that arachidonylethanolamide ("anandamide", AEA), the major endogenous ligand of CB1 receptors, inhibits motor behavior in rats, as does (-)delta 9-tetrahydrocannabinol (THC), the prototypical tricyclic cannabinoid derived from Cannabis sativa preparations. However, its effects were of shorter duration, as compared to THC, likely due to its rapid breakdown by an amidase activity. The present work has been designed to examine the motor effects of AM356(R-methanandamide), an analog of AEA that possesses higher metabolic stability to amidase hydrolysis. We have studied the dose-response and time-course effects of R-methanandamide, i.p. administered, on ambulatory activity, frequency of stereotypy and time spent in inactivity measured in an open-field test. Results were as follows. R-Methanandamide, as THC and AEA, inhibited motor behavior. Thus, it decreased ambulation and stereotypy and increased the time spent in inactivity, usually in a dose-related manner, 10 min after administration. However, the motor deficit caused by the highest dose of R-methanandamide was usually more pronounced than that caused by a similar dose of AEA. These inhibitory effects persisted 30 min after the administration of R-methanandamide, as occurred with AEA and THC. Interestingly, at 60 min after administration, the effects of AEA disappeared, likely because of its breakdown to arachidonic acid and ethanolamine, but this did not occur with R-methanandamide whose effects persisted even until 180 min after treatment as occurred with THC. In summary, R-methanandamide inhibits motor behavior in a manner (its effects were persistent) that resembles the effects of THC rather than the effects of AEA (its effects were of rapid onset but shorter duration). This fact supports the use of R-methanandamide as a valuable tool for studying the physiological roles of the anandamidergic system.

Time-course of the effects of anandamide, the putative endogenous cannabinoid receptor ligand, on extrapyramidal function.

We have recently described the dose-response effect of anandamide (AEA), the N-amide derivative of arachidonic acid that acts as an endogenous ligand for the cannabinoid receptor, on extrapyramidal function. The present study has been designed to examine the time-course of this effect. To this end, adult male rats were submitted to an acute i.p. injection of AEA, delta9-tetrahydrocannabinol (THC) or vehicle and examined at different times after drug administration. Animals were tested in an open-field test, then sacrificed and their striata used for analyses of dopaminergic indices. Results were as follows. The administration of AEA or THC produced the expected inhibition of motor behavior. Thus, the administration of AEA decreased the ambulation and the frequency of stereotypic movements (in particular, the number of rears) and increased the time spent by the rats in inactivity. These effects were evident at 10 and 30 min after the administration of the cannabinoid agonist, but mostly disappeared at 60 min. Interestingly, motor inhibition was observed again around 2 or 3 h after the administration of AEA. This was a small but persistent effect (decreased ambulation followed by increased inactivity), because it was observed until at least 6 h after AEA administration. The other cannabimimetic, THC, was always able of decreasing the ambulation and the frequency of rearing and grooming behavior, and of increasing the time spent in inactivity. This effect was usually something more marked than the effect of AEA, but the most characteristic fact was its persistence at all times studies, even at 6 h after administration. These motor disturbances were accompanied by changes in the activity of nigrostriatal dopaminergic neurons. Thus, the administration of AEA decreased the activity of tyrosine hydroxylase (TH) in the striatum at 10 and 30 min after treatment, suggesting a decreased nigrostriatal activity parallel to the motor deficit observed at these times. This was followed by an increase in TH activity and dopamine and L-3,4-dihydroxyphenylacetic acid contents at 60 min after treatment, which would likely reflect a compensatory stimulation of these neurons, whereas restoration of control values was found at 180 min after AEA administration, suggesting that the motor deficit observed at this time was not dependent on dopaminergic influence. Paradoxically, the administration of THC only produced changes in dopaminergic activity at 60 min after treatment, similar to those seen with AEA, but was ineffective at the other times. In summary, A-EA inhibits motor behavior in parallel to reductions in the activity of nigrostriatal dopaminergic neurons. However, this effect was of short duration, disappearing at 60 min after treatment, as compared with the inhibitory effect of THC on motor behavior which was observed at all times studied. Interestingly, a new AEA-induced inhibition of motor behavior, which was not accompanied by dopaminergic changes, appeared at longer times although its meaning remains to be determined.