Imaging mass spectrometry (IMS) of cortical lipids from preclinical to severe stages of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of patients worldwide. Previous studies have demonstrated alterations in the lipid composition of lipid extracts from plasma and brain samples of AD patients. However, there is no consensus regarding the qualitative and quantitative changes of lipids in brains from AD patients. In addition, the recent developments in imaging mass spectrometry methods are leading to a new stage in the in situ analysis of lipid species in brain tissue slices from human postmortem samples. The present study uses the matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS), permitting the direct anatomical analysis of lipids in postmortem brain sections from AD patients, which are compared with the intensity of the lipid signal in samples from matched subjects with no neurological diseases. The frontal cortex samples from AD patients were classified in three groups based on Braak's histochemical criteria, ranging from non-cognitively impaired patients to those severely affected. The main results indicate a depletion of different sulfatide lipid species from the earliest stages of the disease in both white and gray matter areas of the frontal cortex. Therefore, the decrease in sulfatides in cortical areas could be considered as a marker of the disease, but may also indicate neurochemical modifications related to the pathogenesis of the disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Effects of galanin subchronic treatment on memory and muscarinic receptors.

The cholinergic pathways, which originate in the basal forebrain and are responsible for the control of different cognitive processes including learning and memory, are also regulated by some neuropeptides. One of these neuropeptides, galanin (GAL), is involved in both neurotrophic and neuroprotective actions. The present study has evaluated in rats the effects on cognition induced by a subchronic treatment with GAL by analyzing the passive avoidance response, and the modulation of muscarinic cholinergic receptor densities and activities. [(3)H]-N-methyl-scopolamine, [(3)H]-oxotremorine, and [(3)H]-pirenzepine were used to quantify the density of muscarinic receptors (MRs) and the stimulation of the binding of guanosine 5'-(γ-[(35)S]thio)triphosphate by the muscarinic agonist, carbachol, to determine their functionality. Some cognitive deficits that were induced by the administration of artificial cerebrospinal fluid (aCSF) (i.c.v. aCSF 2 µl/min, once a day for 6 days) were not observed in the animals also treated with GAL (i.c.v. 1.5 mmol in aCSF, 2 µl/min, once a day for 6 days). GAL modulates the changes in M1 and M2 MR densities observed in the rats treated with aCSF, and also increased their activity mediated by G(i/o) proteins in specific areas of the dorsal and ventral hippocampus. The subchronic administration of the vehicle was also accompanied by an increased number of positive fibers and cells for GAL around the cortical tract of the cannula used, but that was not the case in GAL-treated rats. In addition, the increase of GAL receptor density in the ventral hippocampus and entorhinal cortex in the aCSF group was avoided when GAL was administered. The number of acetylcholinesterase (AChE)-positive neurons was decreased in the nucleus basalis of Meynert of both GAL- and aCSF-treated animals. In summary, GAL improves memory-related abilities probably through the modulation of MR density and/or efficacy in hippocampal areas.

Galanin activated Gi/o-proteins in human and rat central nervous systems.

The neuropeptide galanin (GAL) is involved in the control of hormone secretion, nociception, feeding behavior, attention, learning and memory. The anatomical localization of galanin receptors in the brain has been described using autoradiography and immunohistochemistry, but both techniques are limited by the availability of specific radioligands or antibodies. Functional autoradiography provides an alternative method by combining anatomical resolution and information of the activity mediated by G-protein coupled receptors. The present study analyzes the functional GAL receptors coupled to Gi/o-proteins in human and rat brain nuclei using [(35)S]GTPγS autoradiography. The results show the anatomical distribution of Gi/o-proteins activated by GAL receptors that trigger intracellular signaling mechanisms. The activity mediated by GAL receptors in human and rat brain showed a good correlation of the net stimulation in areas such as spinal cord, periaqueductal gray, putamen, CA3 layers of hippocampus, substantia nigra and diverse thalamic nuclei. The functional GAL receptors coupled to Gi/o-proteins showed a similar pattern for both species in most of the areas analyzed, but some discrete nuclei showed differences in the activity mediated by GAL, such as the ventroposteromedial thalamic nucleus, or areas that regulate learning and memory processes in the hippocampus. Taken into consideration the present results, the rat could be used as an experimental model for the study of the physiological role of GAL-mediated neurotransmission and the modulation of GAL receptors activity in the human CNS.

Matrix-assisted laser desorption ionization imaging mass spectrometry in lipidomics.

The relevant structural, energetics, and regulatory roles of lipids are universally acknowledged. However, the high variability of lipid species and the large differences in concentrations make unraveling the role played by the different species in metabolism a titanic task. A recently developed technique, known as imaging mass spectrometry, may shed some light on the field, as it enables precise information to be obtained on the location of lipids in tissues. A review of the state of the art of the technique is presented in this manuscript, including detailed analysis of sample-preparation steps, data handling, and the identification of the species mapped so far.

G protein-coupled galanin receptor distribution in the rat central nervous system.

The action of galanin in the central nervous system is mediated by at least three galanin receptor subtypes (GalR1, GalR2 and GalR3) which belong to the family of G protein-coupled receptors. GalR1 and GalR2 are coupled to G(i/o) proteins, although the latter may also be coupled to G(q/11) proteins. The aim of the present study was to identify the anatomical distribution and quantify the density of GalRs coupled to G proteins. The galanin (10(-6) M) stimulated guanosine 5'-(gamma-[35S] thio)triphosphate binding assay was used in tissue sections from the rat brain. Maximal percentages of stimulation over basal levels were found in the anterior olfactory nucleus and in the lateral olfactory tract nucleus ( approximately 54%). High levels of stimulation were recorded in diverse hypothalamic nuclei (16-28%), in the amygdala (central amygdaloid nucleus, 40%), in the spinal trigeminal tract (23%) and in layers 1-2 of the spinal cord (26%). Moderate binding stimulation (5-13%) was observed in thalamus, substantia nigra pars compacta, parabrachial nucleus, locus coeruleus and dorsal raphe nucleus. The lowest stimulation induced by galanin was recorded in diverse areas of the cortex, striatum, hippocampus and substantia nigra pars reticulata. The results show an anatomical distribution similar to that described for GalR1. However, in diverse brain areas, in which a high density of these receptors has previously been reported, only a moderate coupling to G proteins was found. These findings would suggest that the efficacy of galanin to induce an effective coupling of its receptors to G proteins could be different depending on the brain area.

Effects of central galanin administration on muscarinic cholinergic and galanin receptor G protein coupling.

The neuropeptide galanin is expressed in the mammalian central nervous system and has been implicated in neurotrophic actions. Central galanin administration induces cognitive deficits in rodents and inhibits the release of acetylcholine in the hippocampus. In addition, a galanin hyperinnervation of the basal forebrain cholinergic cells in Alzheimer's disease patients has been reported. To evaluate the effect of galanin treatment on galanin and muscarinic cholinergic receptor G protein coupling, galanin was administered into the lateral ventricle of rats via an implanted cannula. Galanin or muscarinic receptor functional coupling to G proteins was quantified by galanin or carbachol stimulation of guanosine 5'-(gamma-[35S]thio)triphosphate binding in rat brain slices. Guanosine 5'-(gamma-[35S]thio)triphosphate basal binding in nucleus basalis of Meynert and thalamic nuclei was increased in the vehicle treated group. This effect was reverted by galanin treatment and indicates that the surgery increased receptor functional coupling to G proteins, which is restored by a possible neurotrophic action mediated by galanin. In addition, in galanin administered animals, galanin-stimulated binding was increased in the amygdala but decreased in the diagonal band, whilst binding stimulation mediated by carbachol was found to be increased in the amygdala, thalamic nuclei and diagonal band. These findings indicate that galanin treatment modulates the coupling of galanin and muscarinic cholinergic receptors to G proteins in specific regions of the rat central nervous system.

Desensitization of alpha(2)-adrenoceptors which regulate noradrenaline synthesis and release after chronic treatment with clorgyline in the rat brain.

The sensitivity of alpha(2)-adrenoceptors which regulate synthesis and release of noradrenaline was investigated in hippocampus, parietal cortex, and hypothalamus of rats treated with clorgyline. After administering a DOPA decarboxylase inhibitor, the in vivo tyrosine hydroxylase activity and the noradrenaline content were evaluated. Acute and chronic treatment with clorgyline led to both increases of noradrenaline levels and decreases of tyrosine hydroxylase activity, determined as the accumulation of DOPA. Whereas the alpha(2)-adrenoceptor agonist clonidine induced a similar reduction in tyrosine hydroxylase activity in the group subjected to the acute treatment and in the control group, it failed to do so after chronic clorgyline treatment. In hippocampal and cortical synaptosomes, a reduction in the sensitivity of alpha(2)-adrenoceptors which regulate [(3)H]noradrenaline release, reflected by the shift to the right of the concentration-effect curves for oxymetazoline, was also found after the repeated treatment. These results indicate a desensitization of alpha(2)-adrenoceptors after chronic treatment with clorgyline.

Effects of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) on alpha2-adrenoceptors which regulate the synthesis and release of noradrenaline in the rat brain.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) induces a degeneration of noradrenergic axons originating in the locus coeruleus. The sensitivity of alpha2-adrenoceptors which regulate the synthesis and release of noradrenaline was investigated in three brain regions which receive an unequal innervation from locus coeruleus, 21 days after DSP4 (50 mg/kg) administration. After giving treated rats a dopa decarboxylase inhibitor, the in vivo tyrosine hydroxylase activity and the tissue concentrations of noradrenaline were also evaluated. Relevant reductions of noradrenaline levels were found in hippocampus and parietal cortex (91% and 77.5%, respectively; P<0.001) together with a less pronounced reduction in hypothalamus (32%, P<0.01). The administration of the neurotoxin led to decreases of the basal tyrosine hydroxylase activity, determined as the accumulation of 3,4-dihydroxyphenylalanine, in hippocampus and parietal cortex (75% and 50.5%, respectively; P<0.001), but not in hypothalamus. The inhibitory effect of clonidine on tyrosine hydroxylase activity was markedly reduced in hippocampus of rats treated with DSP4 (10+/-5% vs 57+/-3% in the control group, P<0.001) but was not changed in parietal cortex and hypothalamus. Moreover, in hippocampus, a lack of functionality of the alpha2-adrenoceptors which regulate K(+)-evoked [3H]noradrenaline release was determined. However, in cortical synaptosomes the concentration-effect curve for the oxymetazoline shifted to the right. The administration of the neurotoxin did not modify the inhibitory effects of the agonist in hypothalamus. These results support the previously described selectivity of DSP4 for noradrenergic terminals arising from locus coeruleus and suggest a more severe lesioning of the hippocampus than the parietal cortex.

Pilocarpine treatments differentially affect alpha2-adrenoceptors which modulate the firing rate of locus coeruleus neurones and the synthesis and release of noradrenaline in rat brain.

We have investigated the effect of treatments with the muscarinic acetylcholine receptor agonist, pilocarpine, on the sensitivity of central alpha2-adrenoceptors that regulate the firing activity of rat locus coeruleus, the tyrosine hydroxylase activity in the rat cortex, hippocampus and hypothalamus, and the K(+)-evoked release of [3H]noradrenaline from rat cortical and hippocampal synaptosomes. Short-term (4 days), but not acute, treatment with pilocarpine caused a small but statistically significant increase in the inhibitory effect of the alpha2-adrenoceptor agonist clonidine on the firing rate of locus coeruleus neurones, with a decrease in the ED50 of 290% (P<0.001). However, no change in the effect of clonidine on the locus coeruleus was observed after longer pilocarpine (11 days) treatment. In the rat cerebral cortex, but not in hippocampus or hypothalamus, chronic (19 days) treatment with pilocarpine caused a decrease in the inhibitory effect of clonidine on tyrosine hydroxylase activity (55%, P<0.05), but did not change the stimulatory effect of the alpha2-adrenoceptor antagonist idazoxan. Moreover, treatments (4, 11 and 19 days) with pilocarpine did not alter the inhibitory effect of clonidine [10(-8)-10(-5) M] on the K(+)-evoked release of [3H]noradrenaline from rat cortical and hippocampal synaptosomes. These results indicate that administration of pilocarpine slightly potentiates some but not all the functional responses mediated by brain presynaptic alpha2-adrenoceptors. In conclusion, these results do not support the hypothesis that chronic treatments with pilocarpine lead to a suitable model of alpha2-adrenoceptor supersensitivity.

Occlusal disharmonies modulate central catecholaminergic activity in the rat.

Occlusal disharmonies have classically been thought to be involved in the etiopathogenesis of bruxism, as have, more recently, alterations in central neurotransmission, particularly dopaminergic neurotransmission. However, the connection between these two factors has still not been established. In this study, we assessed the effects of diverse occlusal disharmonies, maintained for either 1 day or 14 days, on neurochemical indices of dopaminergic and noradrenergic activity in the striatum, frontal cortex, and hypothalamus of the rat. The in vivo activity of tyrosine hydroxylase, determined as the accumulation of 3,4-dihydroxyphenylalanine (DOPA), 30 min after the administration of 3-hydroxybenzylhydrazine, a DOPA decarboxylase inhibitor, and dopamine and noradrenaline contents were quantified by high-performance liquid chromatography with electrochemical detection. The wearing of an acrylic cap on both lower incisors for 1 day induced a significant increase in DOPA accumulation in the regions analyzed, with parallel increases in dopamine levels in the hypothalamus and dopamine and noradrenaline in the frontal cortex. After the cap was maintained for 14 days, DOPA accumulation tended to return to control values, except in the left striatum, thereby causing an imbalance between hemispheres. In contrast, 1 or 14 days after the lower left and the upper right incisors were cut, less pronounced changes in catecholaminergic neurotransmission were found in the brain areas studied. Moreover, the cutting of one lower incisor did not modify either DOPA accumulation or dopamine and noradrenaline contents in the striatum or hypothalamus. These results provide experimental evidence of a modulation of central catecholaminergic neurotransmission by occlusal disharmonies, being dependent on the nature of the incisal alteration and on the time during which it was maintained.

A possible attenuation of stress-induced increases in striatal dopamine metabolism by the expression of non-functional masticatory activity in the rat.

It is thought that the expression of oral parafunctions may provide an outlet for stress or aggressiveness in man. Stress-induced increases in central noradrenergic neurotransmission are attenuated in rats which are allowed to bite during stress. Striatal dopaminergic neurotransmission is involved in the genesis of parafunctional oral movements in rodents. As tail pinch is the stressor which most clearly provokes non-functional masticatory activity (NFMA), and also increases striatal dopamine (DA) activity in rats, we investigated whether the expression of NFMA during tail pinch could modify the changes in striatal dopaminergic neurotransmission induced by this stressor. Rats were subjected to tail pinch for 5 min, and the duration of the NFMA displayed was recorded. As an index of dopaminergic activity, 3,4-dihydroxyphenylalanine (DOPA) accumulation and DA and 3,4-dihydroxyphenylacetic (DOPAC) contents in both striata were determined by high-performance liquid chromatography. Striatal DOPA accumulation was similarly increased in relation to control, both in rats which did and did not display NFMA during tail pinch. However, the increases in striatal DOPAC contents, reported 24 min after the stress session, were lower in animals which had displayed NFMA. These results provide further evidence in support of the assumption that the expression of parafunctional masticatory activity attenuates the effects of stress on central catecholaminergic neurotransmission.

Chronic variable stress induces supersensitivity of central alpha2-adrenoceptors which modulate the jaw-opening reflex in the rat.

In a previous study, we found that the sensitivity of central postsynaptic alpha2-adrenoceptors which modulate, in an inhibitory way, the activity of the jaw-opening reflex (JOR) is reduced after chronic repeated stress (tail pinch) in the rat. The aim of this study was to assess the effects of exposure to a chronic variable stress regime on these adrenoceptors. To do this, the digastric electromyographic responses elicited by orofacial electrical stimulation after the intravenous administration of cumulative doses (x3.3) of the alpha2-adrenoceptor agonist, clonidine (0.1-10000 microgram/kg), were recorded. As expected, in unmanipulated control rats, clonidine inhibited the reflex, in a dose-dependent manner, until abolition (ED50 = 17.3 +/- 2.2 microgram/kg). Single tail pinch did not significantly alter the ability of clonidine to abolish the reflex. However, chronic variable stress led to an enhancement of the inhibitory effect of clonidine on the amplitude of JOR, resulting in a shift to the left of the dose-response curve in comparison with that of the control group (ED50 was reduced by 37%, P = 0.032), without affecting either the estimated maximum effect for the agonist or the slope of the inhibitory function. This in vivo result indicates that chronic variable stress leads to an increased sensitivity of central alpha2-adrenoceptors which modulate JOR, in contrast to the desensitization of these adrenoceptors found after repeated exposure to the same stressor.

Effects of dopaminergic drugs, occlusal disharmonies, and chronic stress on non-functional masticatory activity in the rat, assessed by incisal attrition.

Observational methods and the recording of nonspecific jaw movements or masticatory muscle activity have been used to evaluate oral parafunctional movements in animal models of bruxism. In this study, we have used a new approach in which the non-functional masticatory activity in the rat was assessed by the measurement of incisal attrition, with the aim of investigating the role of diverse factors involved in the etiology of bruxism. We quantified the attrition rate weekly by making superficial notches in the lower incisors and measuring the distances to the incisor edges. Repeated stimulation of the dopaminergic system with apomorphine led to an enhancement of the non-functional masticatory activity (p < 0.0001). The severity of the apomorphine-induced oral behavior was positively correlated (r(s) = 0.69, p < 0.01) with an increase in the incisal attrition rate (20.9%, p < 0.0001). Apomorphine-induced non-functional masticatory activity was strongly enhanced by the placement of an acrylic cap on both lower incisors (306%, p < 0.0001), but not by the cutting of a lower incisor. Repeated cocaine administration also increased the attrition rate (22.5%, p < 0.0001). However, neither chronic blockade of dopaminergic receptors with haloperidol, nor its withdrawal, modified attrition. In addition, since emotional disturbances are considered to be causal factors of bruxism, we tested whether experimental stress might accelerate tooth wear. Exposure to two different chronic stress regimes did not induce significant changes in incisal attrition. Moreover, exposure to chronic stress after the withdrawal of chronic haloperidol treatment did not alter attrition either. These results partially support the role of the central dopaminergic system in bruxism and suggest that stress, in general, may not be a relevant factor in tooth wear.

Acute and chronic effects of reserpine on biochemical and functional parameters of central and peripheral alpha 2-adrenoceptors.

The specific binding of the agonist, [3H]UK 14304, and of the antagonist, [3H]RX 821002, to rat brain membranes, as well as clonidine-induced mydriasis, clonidine-induced inhibition and idazoxan-induced stimulation of brain 3,4-dihydroxyphenylalanine (DOPA) synthesis, and clonidine and UK 14304-induced inhibition of twitch responses in the vas deferens were used to evaluate the affinity and sensitivity of central and peripheral alpha 2-adrenoceptors after various treatments with reserpine. Treatment with reserpine (0.25 mg/kg s.c., every 48 h) for 4, 11 and 18 days induced consistent and significant increases in the affinity (KD values) of [3H]UK 14304 for the cortical alpha 2-adrenoceptor with no change in receptor density. Chronic treatment with reserpine also resulted in a greater affinity of (-)-adrenaline for the high-affinity state of the alpha 2-adrenoceptor when the catecholamine competed with the binding of [3H]RX 821002 to cortical membranes. In line with these radioligand binding data, various functional responses mediated by central and peripheral alpha 2-adrenoceptors were found to be potentiated after repeated treatment with reserpine. Thus, the inhibitory alpha 2-autoreceptor that modulates the synthesis of brain noradrenaline and the central postsynaptic inhibitory alpha 2-adrenoceptor that induces mydriasis displayed greater responses in vivo after chronic treatment with reserpine. Short-term and chronic treatments with reserpine also increased the sensitivity of peripheral presynaptic alpha 2-adrenoceptors in the vas deferens.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute and chronic effects of cholinesterase inhibitors and pilocarpine on the density and sensitivity of central and peripheral alpha 2-adrenoceptors.

The specific binding of the agonists [3H]clonidine and [3H]UK 14304 (bromoxidine) and of the antagonist [3H]RX 821002 (2-metoxy idazoxan) to rat brain membranes, as well as clonidine-induced mydriasis, clonidine-induced inhibition of brain (3,4-dihydroxyphenylalaninme) DOPA synthesis and clonidine-induced inhibition of twitch responses in the vas deferens, was used to evaluate the density and sensitivity of central and peripheral alpha 2-adrenoceptors after prolonged activation of the cholinergic system. Acute (12 h), short-term (4 days) or chronic (7-18 days) treatment with the cholinesterase inhibitors neostigmine (0.1 mg/kg), physostigmine (0.1 mg/kg) and diisopropylfluorophosphate (2 mg/kg) and with the muscarinic receptor agonist pilocarpine (10 mg/kg) did not alter the density of brain alpha 2-adrenoceptors. In contrast, various functional responses mediated by central and peripheral alpha 2-adrenoceptors were potentiated after the repeated treatments. Thus, the inhibitory alpha 2-autoreceptor that modulates the synthesis of brain noradrenaline and the central postsynaptic inhibitory alpha 2-adrenoceptor that induces mydriasis displayed greater responses in vivo after chronic treatment with neostigmine or pilocarpine. These treatments also increased the sensitivity of peripheral presynaptic alpha 2-adrenoceptors in the vas deferens. The results indicate that prolonged activation of central and peripheral cholinergic pathways results in up-regulation of alpha 2-adrenoceptor function without apparent increases in receptor density.

Alpha 2-adrenoceptor-mediated inhibition of platelet adenylate cyclase and induction of aggregation in major depression. Effect of long-term cyclic antidepressant drug treatment.

The functional status of platelet alpha 2-adrenoceptors in patients with major depression has been assessed by simultaneously measuring both a biochemical mechanism of transduction of receptor activation (inhibition of adenylate cyclase activity) and a physiologic response of the receptor (induction of aggregation). The inhibitory effects induced by epinephrine and UK 14304 on adenylate cyclase activity were unchanged, while the aggregation responses induced by the same alpha 2-adrenoceptor agonists were potentiated, which indicated receptor supersensitivity. In depressed (n = 30) and euthymic (n = 11) patients as well as in control subjects (n = 66), there was a clear dissociation between inhibition of adenylate cyclase activity and induction of aggregation, indicating that the two responses represent different phenomena of alpha 2-adrenoceptor activation. alpha 2-Adrenoceptor-mediated platelet aggregation could represent a better marker than inhibition of adenylate cyclase to assess functional changes of the receptor in depression. Both of these functional responses are desensitized after long-term antidepressant treatment.

GABA terminal autoreceptors in the pars compacta and in the pars reticulata of the rat substantia nigra are GABAB.

The depolarization-evoked release of gamma-aminobutyric acid (GABA) and its modulation mediated by autoreceptors were studied in superfused synaptosomes prepared from the pars compacta and from the pars reticulata of the rat substantia nigra. The release of [3H]GABA evoked by 9 mM KCl was almost totally calcium-dependent in both nigral subregions. In the presence of SK&F 89976A (N-(4,4-diphenyl-3-butenyl)nipecotic acid), a GABA uptake inhibitor added to minimize carrier-mediated homoexchange, GABA (0.3-10 microM) inhibited, in a concentration-dependent way, the K(+)-evoked overflow of [3H]GABA from both pars compacta and pars reticulata synaptosomes. Similarly to GABA, (-)-baclofen (0.3-10 microM) reduced the [3H]GABA overflow, being roughly equipotent to GABA in both nigral subregions. The (+) enantiomer of baclofen was ineffective. The overflow of [3H]GABA was not consistently affected by muscimol in either the pars compacta or the pars reticulata. The effects of GABA were bicuculline- and picrotoxin-insensitive. However, the inhibition by GABA of the [3H]GABA overflow was antagonized by phaclofen. It is concluded that (a) GABA autoreceptors are sited on GABAergic nerve endings in both the pars compacta and pars reticulata of the rat substantia nigra; (b) these autoreceptors belong to the GABAB type.

Acute and long-term regulation of brain alpha 2-adrenoceptors after manipulation of noradrenergic transmission in the rat.

The specific binding of [3H]clonidine (KD and Bmax) to rat brain membranes was used as a biochemical index to directly evaluate alpha 2-adrenoceptor changes after manipulation of synaptic noradrenaline (NA) pools or stimulation or blockade of the receptor. Acute (2 h) and prolonged (7 days) inhibition of NA synthesis with alpha-methyl-p-tyrosine (150 mg/kg) or acute (2 h) and chronic (14 days) treatment with reserpine (0.1-0.5 mg/kg) reduced the NA content by 15-90%, which also resulted in marked reductions (35-55%) of the KD values for [3H]clonidine in all brain regions studied. In contrast to alpha-methyl-p-tyrosine, chronic reserpine treatment did not alter the Bmax values for [3H]clonidine or [3H]UK 14304 in any brain region. In the hypothalamus and cerebral cortex, acute (2 h) and chronic (7-14 days) treatment with the monoamine oxidase (MAO) inhibitors clorgyline (1 mg/kg) or tranylcypromine (5 mg/kg) increased the content of NA by 6-100%, which led to marked reductions (20-50%) of Bmax without altering the KD values for [3H]clonidine. Similarly, prolonged (21 days) inhibition of NA neuronal uptake with cocaine or protriptyline (10 mg/kg) also resulted in decreases in Bmax (20-25%) with no alterations in KD in the hypothalamus. In various brain regions, chronic (14 days) but not short-term (1 day) treatment with clonidine (0.1 mg/kg) or yohimbine (10 mg/kg) resulted in decreases (30-40%) and increases (15-20%), respectively, in Bmax without altering the KD values for [3H]clonidine. The results indicate that drugs which deplete endogenous NA up-regulate alpha 2-adrenoceptors (increased affinity of [3H]clonidine binding sites) while drugs which increase the intraneuronal and/or synaptic NA pools down-regulate the receptors (decreased number of [3H]clonidine binding sites). These adaptive receptor changes appear to be dependent on NA availability.

Chronic naltrexone suppresses platelet aggregation induced by adrenaline and 5-hydroxytryptamine in former heroin addicts.

Naltrexone, an opioid receptor antagonist, is used as an adjunct in the treatment of opiate addiction. In former heroin addicts, long-term treatment with naltrexone (350 mg/week for 5 months) resulted in suppression of adrenaline and 5-hydroxytryptamine (5-HT)-induced platelet aggregation. The results demonstrate that sustained blockade of opioid receptors can impair the functional expression of alpha 2-adrenoceptors and 5-HT2 receptors in human platelets. These findings may have negative clinical implications in the treatment of opiate addiction with naltrexone.

Uncoupling of the platelet alpha 2-adrenoceptor adenylate cyclase system by N-ethylmaleimide and the resulting effect on receptor density and sensitivity.

Preincubation of platelet-rich plasma with N-ethylmaleimide (NEM) attenuated the inhibitory effect of the alpha 2-adrenoceptor agonist UK 14304 on basal and forskolin-stimulated adenylate cyclase activities. NEM also led to concomitant marked reductions of the specific binding of [3H]UK 14304 to platelet membranes and of the primary aggregation response induced by UK 14304. These results indicate that uncoupling of the receptor adenylate cyclase system by NEM induces down-regulation of platelet alpha 2-adrenoceptor density (3H-agonist binding sites) and of the associated functional response (platelet aggregation).