Neuroprotective effect of N-acylethanolamines in chronic morphine dependence. II. Effect on the rat brain fatty acid composition

The effect of N-acylethanolamines mixture (NAE) with saturated and unsaturated acyl chains on the fatty acid composition of the rat brain under chronic morphine dependence was investigated. Long-term administration of NAE reduced morphine-induced decrease of mono- and polyunsaturated fatty acids in the rat brain crude lipid extract. Furthermore, NAE restored the acyl chain spectrum, especially the content of docosahexaenoic acid in essential phospholipids--phosphatidylcholine and phosphatidylethanolamine. Pharmacological activity of NAE depended on a dose.     

Neuroprotective effect of N-acylethanolamines in chronic morphine dependence. III. Influence on the content of neurotransmitters in the rat brain

The effect of the mixture of saturated and unsaturated N-acylethanolamines (NAEs) on the functional activity of catecholamine- and serotoninergic systems of the rat brain with experimental morphine dependence was investigated. A significant decrease of dopamine levels was found in the hypothalamus, midbrain and cortex of rats with morphine dependence. The administration of NAEs to rats with morphine dependence in time course dose of 35 mg/kg markedly increased the levels of dopamine in the hypothalamus, middle brain and cortex. Simultaneously the significant decrease of serotonine content was observed in the midbrain and cortex. The results obtained suggest that one of the important aspects of neuroprotective action of NAEs under morphine dependence is the restoration of dopamine content in the brain.     

Rat brain proteome in morphine dependence

The aim of this study was to reveal potential markers associated with drug dependence, using the proteomic approach. Gels containing samples derived from morphine-treated and control animals were compared and analyzed. Inspection of protein profiles, following TCA/acetone precipitation and the use of nano-scale liquid chromatography coupled to tandem mass spectrometry, allowed for identification of eleven potential dependence markers, mainly cytoplasmic and mitochondrial enzymes, e.g. proteins that belong to GTPase and GST superfamilies, ATPase, asparaginase or proteasome subunit p27 families.     

A candidate target for G protein action in brain.

An effector candidate for G protein action, GRIN1, was identified by screening a cDNA expression library with phosphorylated GTPgammaS-G(z)alpha as a probe. GRIN1 is a novel protein without substantial homology to known protein domains. It is expressed largely in brain and binds specifically to activated G(z)alpha, G(o)alpha, and G(i)alpha through its carboxyl-terminal region. The protein KIAA0514 (GRIN2) is homologous to GRIN1 at its carboxyl terminus and also binds to activated G(o)alpha. Both GRIN1 and G(o)alpha are membrane-bound proteins that are enriched in the growth cones of neurites. Coexpression of GRIN1 or GRIN2 with activated G(o)alpha causes formation of a network of fine processes in Neuro2a cells, suggesting that these pathways may function downstream of G(o)alpha to control growth of neurites.     

Hippocampus: a target for estrogen action in mammalian brain

An analysis of the distribution of estrogen receptor (ER) via immunoenzymatic assay in the brain of ovariectomized rats reveals the presence of large amounts of ER-like immunoreactive material in the cytosol of the hippocampus: a brain area described to contain little estrogen-binding activity. The protein detected in the hippocampus by the specific antibody is indistinguishable from the rat ER in its response to hormonal treatments and in its electrophoretic mobility. The presence of elevated amounts of ER in such an important part of the limbic system creates new possibilities for interpreting the role played by this sex hormone in the central nervous system of rat.     

Evidence of possible opiate dependence during the behavioral depressant action of a single dose of morphine

Rats were trained to lever press for food pellets under a 20 response fixed ratio (FR 20) schedule of reinforcement. A single injection of 15 mg morphine SO₄/kg suppressed operant behavior for $\text{1}\text{1}\text{2}\text{–3}\text{1}\text{2}\text{hrs}$, after which time responding resumed at a reduced rate. When 0.25 mg naloxone HCl/kg was given during the recovery phase, the behavioral depressant effect of the narcotic was immediately reversed and operant performance returned to predrug rates. In contrast, when 0.5 mg naloxone/kg was given at this time, operant behavior was abolished for at least 1 hr. Naloxone, at these doses, did not affect responding in drug-naive subjects. These results suggest that a single, relatively low dose of morphine can induce transient dependence which is detectable for several hrs after drug administration, at a time when the acute pharmacological actions of morphine are still apparent.     

Digoxin Exhibits Neuroprotective Properties in a Rat Model of Dementia

Neurochemical Research - Alzheimer's disease (AD) is by far the most common cause of cognitive impairment in older adults. Current treatments are entirely focused on the symptoms of AD. A...     

Rat brain polysomal profiles: a study of their stability in vitro

M odifications of the profiles of brain polysomes have been interpreted as having pathological significance (A oki and S iegel , 1970; V esco and G iuditta , 1968). However, profiles of polysomes obtained from brains of healthy animals vary greatly with the method of extraction (R oberts and Z omzely , 1966) and we do not know whether they accurately reflect the distribution in oioo of polysomes and ribosomal subunits. In exponentially growing E. coli, the large proportion of 70s ribosomes found in polysomal profiles is an artifact resulting from the aggregation in vitro of ribosomal subunits (P hillips , H otham -I glewski and F ranklin , 1969a, b). In the present study we sought to determine whether similar changes reflecting instability of some components or in vitro aggregation are likely to occur in polysomes from rat brain. Initially we have investigated the effects of various monovalent cations on the profiles of brain polysomes and the aggregation in vitro of ribosomal subunits.     

Inhibition of morphine dependence by a lipopolysaccharide from Pantoea agglomerans.

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified and examined for relief of morphine dependence by observing its inhibition of the jumping of mice on naloxone-precipitate withdrawal. Administration of LPSp either intravenously or intradermally showed marked inhibition of the jumping. Beta-endorphin in mouse serum and brain tissue were recognized to be in synchrony with the time course of the relief. Administration of TNF-alpha gave similar effect, suggesting that LPSp induces a cytokine cascade to produce endogenous TNF followed by ACTH/beta-LPH gene products and beta-endorphin. The effect of LPSp was better than that of LPS from E. coli or Bordetella pertussis, and thus is considered to be applicable for clinical use.     

Rat choroidal pericytes as a target of the autonomic nervous system

Pericytes are contractile cells that surround blood vessels. When contracting, they change the diameter of the vessel and therefore influence blood flow homeostasis; however, mechanisms controlling pericyte action are less well understood. Since blood flow regulation per se is controlled by the autonomic nervous system, the latter might also be involved in pericyte action. Hence, rat choroidal pericytes were analyzed for such a connection by using appropriate markers. Rat choroidal wholemounts and sections were prepared for immunohistochemistry of the pericyte marker chondroitin-sulfate-proteoglycan (NG2) and the pan-neuronal marker PGP9.5 or of tyrosine hydroxylase (TH), vasoactive intestinal polypeptide (VIP) and choline acetyl transferase (ChAT). Additionally, PGP9.5 and TH were analyzed in the choroid of DCX-dsRed2 transgenic rats, displaying red-fluorescent perivascular cells and serving as a putative model for studying pericyte function in vivo. Confocal laser-scanning microscopy revealed NG2-immunoreactive cells and processes surrounding the blood vessels. These NG2-positive cells were not co-localized with PGP9.5 but received close appositions of PGP9.5-, TH-, VIP- and ChAT-immunoreactive boutons and fibers. In the DCX-dsRed2 transgenic rat, PGP9.5 and TH were also densely apposed on the dsRed-positive cells adjacent to blood vessels. These cells were likewise immunoreactive for NG2, suggesting their pericyte identity. In addition to the innervation of vascular smooth muscle cells, the close relationship of PGP9.5 and further sympathetic (TH) and parasympathetic (VIP, ChAT) nerve fibers on NG2-positive pericytes indicated an additional target of the autonomic nervous system for choroidal blood flow regulation. Similar findings in the DCX-dsRed transgenic rat indicate the potential use of this animal model for in vivo experiments revealing the role of pericytes in blood flow regulation.     

BTX modification of Na channels in squid axons. I. State dependence of BTX action

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.     

The effect of mepyramine on the development of morphine tolerance and physical dependence in mice.

Latency relaxation (LR) as well as resting tension and twitch tension of frog toe muscles are studied in an isotonic solution (= 1 T) and in solutions made hypotonic by leaving out the appropriate amounts of NaCl and KCl (0.54 T and 0.76 T). In hypotonic solutions there is an increase in peak twitch tension as well as a decrease in the depth of the LR: the resting tension is increased at sarcomere lengths which are greater than 2.8 μm and is decreased at sarcomere lengths which are less than this value. The behaviour of twitch tension is discussed with respect to the influence of the sarcoplasmic ionic strength on the interaction between the contractile filaments. Concerning the decrease in both the LR and the resting tension, it is assumed that these effects are induced osmotically, the tension of the membranes of the longitudinal sarcoplasmic reticulum being the particular parameter which is influenced.     

Neuroprotective effect of Danshensu derivatives as anti-ischaemia agents on SH-SY5Y cells and rat brain

Novel Danshensu derivatives (3–8) were designed and synthesized to improve bioactivity based on the strategy of ‘medicinal chemical hybridization’. Our previous studies indicated that these compounds exhibited noticeable cardioprotective activities. Here, we investigate whether these novel Danshensu derivatives exert neuroprotective activities. An in vitro study revealed that these compounds could increase cell viability and reduce LDH (lactate dehydrogenase) leakage. Moreover, Danshensu-cysteine derivative compounds 6 and 8 could significantly inhibit lipid peroxidation of cell membrane and regulate the expression of apoptosis-related protein (Bcl-2, Bax and caspase 3). An in vivo study demonstrated that treatment with compound 6 at 30 mg/kg markedly decreased the infarct volume of MCAO (middle cerebral artery occlusion) insulted rat brain. Furthermore, treatment with compound 6 showed the antioxidant capacity by increasing the activity of SOD (superoxide dismutase) and GPx (glutathione peroxidase) and decreasing the level of MDA (malondialdehyde) and the ROS (reactive oxygen species) production significantly. These results suggested that these novel conjugates exert significant neuroprotective effects as anti-ischaemia agents and those with high potential merit further investigation.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain identification of phospholipids as calcium-independent activators.

Phosphatidyl inositol and lysophosphatidyl choline have been identified as activators of a partially purified brain cyclic nucleotide phosphodiesterase previously shown to be regulated in vitro by Ca²⁺ and a Ca²⁺-binding protein. Microgram quantities of either phospholipid produced a linear, immediate and reversible activation of the enzyme in the absence of Ca²⁺ and the Ca²⁺-dependent regulator (CDR). Fatty acids were also found to activate the phosphodiesterase to varying degrees, with oleic acid being the most effective. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and lysophosphatidyl ethanolamine were not effective as activators. Only sodium dodecyl sulfate, of a variety of nonionic, cationic, and anionic detergents tested, activated the phosphodiesterase. Sodium dodecyl sulfate produced a modest degree of activation over a narrow concentration range, followed by enzyme denaturation at higher concentrations.The interaction of the phosphodiesterase with the phospholipid activators has been compared to its interaction with the Ca²⁺·CDR complex. Both Ca²⁺·CDR and lysophosphatidyl choline decreased the thermal stability of the enzyme to a similar extent. The apparent K_m of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 30 μm with guanosine-3′,5′-monophosphate (cGMP) as substrate and 1 mm with adenosine-3′,5′-monophosphate (cAMP) as substrate. With increasing lysophosphatidyl choline concentration, the apparent K_m for each nucleotide remained unchanged while the V increased. The apparent K_d for Mg²⁺ of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 3 μm and was unaffected by lysophosphatidyl choline concentration. Activation of the phosphodiesterase by lysophosphatidyl choline was characterized by a high degree of positive cooperativity, exhibiting a Hill coefficient of 3.8. Fluphenazine was a competitive inhibitor of both Ca²⁺·CDR and lysophosphatidyl choline activation of the enzyme.     

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on mulitlipid systems

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.     

The aging brain: protein phosphorylation as a target of changes in neuronal function.

There is evidence that senescence affects neurotransmission at different levels. In particular, this review summarizes the studies on age-dependent modifications in protein phosphorylation, which represents the final pathway in the action of transmitters and hormones at neuronal level. Cyclic AMP-dependent protein kinase and protein kinase C have been reported to be modified during aging in various cerebral areas; the changes may involve either enzyme activity or substrate availability. These findings can be related to the alterations in neurotransmitter function and synaptic efficiency observed in the senescent brain. The activity of the other types of protein kinases (tyrosine-, cGMP-, calcium/calmodulin-dependent) during aging needs to be explored. An emerging point is the role of protein phosphorylation in the transfer of membrane signals to the nucleus, for the activation or disactivation of specific genes responsible for long-term neuronal events. Along this view, alterations in protein kinase pathway during senescence would ultimately affect gene expression, resulting in long term modifications of cell function. The reviewed literature opens the perspective of restoring some of the deficits associated with senescence by modulating protein phosphorylation pathway.     

In-vitro evaluation of morphine dependence. I. Response of the rat "fundus" to ACh]

The kinetic of cholinergic receptor systems was studied in an in vitro model for opiate tolerance and dependence. The fundus strip of rats chronically treated with morphine and sacrificed at different time intervals and in different conditions (with and without abstinence signs) was used. In evaluating the responses of fundus to ACh, the cholinergic receptor system showed the same response both in a condition of abstinence and in a condition of non-abstinence in vivo and in vitro. The response does vary however, according to the duration of the treatment: after an initial increase it comes back to its normal values, thus reproducing the tolerance pattern.