[Glu2]TRH dose-dependently attenuates TRH-evoked analeptic effect in mice.

Thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH(2)) and the structurally related [Glu(2)]TRH (pGlu-Glu-Pro-NH(2)) are endogenous peptides with a plethora of actions in the central nervous system. Many centrally-mediated effects of TRH are shared with those of [Glu(2)]TRH, although the involvement of different receptors is presumed. The analeptic action is the best-known TRH-related central nervous system effect. While [Glu(2)]TRH itself is analeptic, its co-administration with TRH into mice produced a dose-dependent attenuation of TRH-evoked reversal of barbiturate-induced sleeping time. This finding is in agreement with our previous observations that [Glu(2)]TRH significantly attenuates TRH-induced hippocampal extracellular acetylcholine release. Taken together, [Glu(2)]TRH may be considered as a negative modulator for the cholinergic effect of TRH in the mouse brain.

Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel.

Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.

Evidence for interplay between thyrotropin-releasing hormone (TRH) and its structural analogue pGlu-Glu-Pro-NH2 ([Glu2]TRH) in the brain: an in vivo microdialysis study.

Local perfusion of pGlu-Glu-Pro-NH2, an endogenous peptide structurally related to thyrotropine-releasing hormone (TRH), via in vivo microdialysis into the rat hippocampus did not change the basal level of extracellular acetylcholine. However, co-perfusion of pGlu-Glu-Pro-NH2 with TRH in equimolar concentrations yielded a significant attenuation of TRH-induced acetylcholine release. The results have supported the study's hypothesis that pGlu-Glu-Pro-NH2 opposes the cholinergic effect of TRH in the mammalian central nervous system. The enantiomer pGlu-d-Glu-Pro-NH2 affected neither basal extracellular nor TRH-induced increase of acetylcholine levels.

Cardiovascular effects of neuropeptide FF antagonists.

The neuropeptide FF (NPFF) antagonist desaminotyrosyl-Phe-Leu-Phe-Gln-Pro-Gln-Arg-NH2 dose-dependently reversed NPFF-induced elevation of blood pressure in anesthetized rats after intravenous injection without causing a significant change of blood pressure and heart rate by itself. However, another antagonist dansyl-Pro-Gln-Arg-NH2 produced a significant drop of the mean arterial pressure only at a large dose (10 micromol/kg body weight), but reversal of the NPFF-induced hypertension was modest. Consequently and contrary to the conclusions of a previous study, NPFF antagonists cannot be identified simply by measuring the changes in the hemodynamic parameters upon the injection of the compounds alone and without a subsequent NPFF challenge.

Effect of chronic morphine exposure on the synaptic plasma-membrane subproteome of rats: a quantitative protein profiling study based on isotope-coded affinity tags and liquid chromatography/mass spectrometry.

The effect of chronic morphine exposure on the synaptic plasma-membrane subproteome in rats was studied by the isotope-coded affinity tag (ICAT) method coupled with capillary reversed-phase liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry. ICAT-labeled tryptic peptides of synaptic membrane proteins were successfully identified using tandem mass spectrometry in conjunction with protein database searching. Several important synaptic plasma-membrane proteins displayed significant regulation changes as a result of chronic morphine exposure in vivo. In particular, an integral membrane protein Na(+)/K+ ATPase (alpha-subunit) involved in regulation of the cell membrane potential by controlling sodium and potassium ion permeability was downregulated by 39 +/- 2%. This result was in excellent agreement with the reduction in electrogenic Na+, K+ pumping due to about 40% downregulation of Na(+)/K+ ATPase alpha3-isoform in myenteric S-neurons of morphine-exposed guinea-pigs measured by others via immunohistochemistry. The decrease in the abundance of non-erythroid alpha II-spectrin in the synaptic plasma-membrane fraction was also observed, which was hypothetically associated with the breakdown of the protein due to the upregulation of the proteolytic enzyme caspase-3 upon chronic morphine exposure.

Centrally acting and metabolically stable thyrotropin-releasing hormone analogues by replacement of histidine with substituted pyridinium.

Metabolically stable and centrally acting thyrotropin-releasing hormone (TRH) analogues were designed by replacing the central histidine with substituted pyridinium moieties. Their analeptic and acetylcholine-releasing actions were evaluated to assess their potency as central nervous system (CNS) agents. A strong experimental connection between these two CNS-mediated actions of the TRH analogues was obtained in subject animals. The analogue 3-(aminocarbonyl)-1-(3-[2-(aminocarbonyl)pyrrolidin-1-yl]-3-oxo-2-[[(5-oxopyrrolidin-2-yl)carbonyl]amino]propyl)pyridinium (1a) showed the highest (TRH-equivalent) potency and longest, dose-dependent duration of action from a series of homologous compounds in antagonizing pentobarbital-induced narcosis when administered intravenously in its CNS-permeable prodrug form (2a) obtained via reduction of the pyridinium moiety to the nonionic dihydropyridine. The maximum change in hippocampal acetylcholine concentration upon perfusion of the pyridinium-containing tripeptides into the hippocampus of rats was also achieved with 1a. No binding to the endocrine TRH receptor was measured for the TRH analogues reported here; therefore, our design afforded a novel lead for centrally acting TRH analogues. We have also demonstrated the benefits of the prodrug approach on the pharmacokinetics and brain uptake/retention of pyridinium-containing TRH analogues (measured by in vivo microdialysis sampling) upon systemic administration.

Proteomic analysis of the synaptic plasma membrane fraction isolated from rat forebrain.

Mass spectrometry (MS) in conjunction with liquid chromatography and gel separation techniques has been utilized to identify synaptic plasma membrane (SPM) proteins isolated from rat forebrain and digested with the protease trypsin. Initial results employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation of the SPM protein mixture have shown that several membrane proteins were under-represented due to solubilization problems in the dimension of isoelectric-point focusing. Given the complexity of the SPM, multiple stages of separation were necessary prior to mass spectrometric detection in order to facilitate protein identification. This particular study involved several approaches using one-dimensional (1D) sodium dodecyl sulfate (SDS)-PAGE, strong cation-exchange (SCX) chromatography and capillary reversed-phase high performance liquid chromatography (HPLC) techniques. In addition to these gel and HPLC separation stages, complementary information was obtained by using both matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. Data-dependent acquisition employing capillary HPLC-nanoESI/MS allowed for the detection of low-abundance tryptic peptides in the digested SPM fraction and identification of the corresponding proteins when product-ion information of a single or multiple peptides was used in protein database searching. The potential value of this subproteome methodology was exemplified by the identification of several proteins relevant to synaptic physiology which included various transporters, receptors, ion channels, and enzymes.

Quinol-based cyclic antioxidant mechanism in estrogen neuroprotection.

Substantial evidence now exists that intrinsic free-radical scavenging contributes to the receptor-independent neuroprotective effects of estrogens. This activity is inherently associated with the presence of a phenolic A-ring in the steroid. We report a previously unrecognized antioxidant cycle that maintains the "chemical shield" raised by estrogens against the most harmful reactive oxygen species, the hydroxyl radical (*OH) produced by the Fenton reaction. In this cycle, the capture of *OH was shown to produce a nonphenolic quinol with no affinity to the estrogen receptors. This quinol is then rapidly converted back to the parent estrogen via an enzyme-catalyzed reduction by using NAD(P)H as a coenzyme (reductant) and, unlike redox cycling of catechol estrogens, without the production of reactive oxygen species. Due to this process, protection of neuronal cells against oxidative stress is also possible by quinols that essentially act as prodrugs for the active hormone. We have shown that the quinol obtained from a 17beta-estradiol derivative was, indeed, able to attenuate glutamate-induced oxidative stress in cultured hippocampus-derived HT-22 cells. Estrone quinol was also equipotent with its parent estrogen in reducing lesion volume in ovariectomized rats after transient middle carotid artery occlusion followed by a 24-h reperfusion. These findings may establish the foundation for a rational design of neuroprotective antioxidants focusing on steroidal quinols as unique molecular leads.

Quinol-based metabolic cycle for estrogens in rat liver microsomes.

According to a recently reported metabolic pathway, phenolic A-ring estrogens are metabolized in rat liver microsomes partially to the corresponding quinols by cytochrome P450 isoenzymes. We found that these quinols could, in turn, undergo reduction to regenerate the parent estrogens consumed during the metabolic process. Among the tested endogenous reducing agents, NADH and especially NADPH produced a significant extent of reductive conversion. Enzymes available in rat liver microsomes further catalyzed this reaction with 6.5 +/- 1.5 nmol. min(-1). (mg of protein)(-1) measured as the initial rate of estrone formation at 37 degrees C, whereas the initial rate of second-order reaction for the reduction of E1-quinol by a 10-fold excess of NADPH in a microsome-free buffer solution and under identical incubation conditions was 0.62 +/- 0.03 nmol. min(-1). The quinol route is, therefore, unique among estrogen-metabolizing pathways for its bioreversibility due to the facile regeneration of the phenolic A-ring estrogens consumed in the preceding oxidative process.

Determination of clodronate content in liposomal formulation by capillary zone electrophoresis.

Liposome-mediated intracellular delivery of clodronate is reported to selectively deplete mononuclear phagocytic cells such as macrophages that are important effector cells involved in the pathogenesis of neuropathies associated with demyelination and destruction of neuronal cells. Application of liposome-encapsulated clodronate (dichloromethylenediphosphonic acid disodium salt) is a method of choice to deplete macrophages to prevent such a neurodegeneration. In the present work, a comparison of an ion-exchange chromatography (IEC) and a capillary zone electrophoresis (CZE) method with indirect UV detection was performed and, based on the results of this comparison, a CZE assay was developed for quantitation of clodronate in mannosylated liposomes. This CZE method employed Nitroso-R salt (1-nitroso-2-naphthol-3,6-disulphonic acid disodium salt) as background electrolyte with UV detection of the analyte at 254 nm. The assay for the determination of clodronate in mannosylated liposomes after their solubilization in 10 mM Triton X-100 showed acceptable within-day precision (repeatability), day-to-day precision (reproducibility) and linearity in the target quantitation range of 0.5-10.0 mg ml(-1). The method reported here can be used as part of the quality control during the preparation of liposome-encapsulated clodronate as a drug formulation for macrophage-mediated diseases.

Prodrugs to enhance central nervous system effects of the TRH-like peptide pGlu-Glu-Pro-NH2.

Potential prodrugs for the TRH-like tripeptide pGlu-Glu-Pro-NH(2) were synthesized either by esterifying the Glu side-chain of the parent peptide in solution with alcohols in the presence of resin-bound dicyclohexylcarbodiimide or by solid-phase peptide chemistry. Affinities of these ester prodrugs to lipid membranes as predictors of the transport across the blood-brain barrier were compared by immobilized artificial membrane chromatography, and prodrug activation was tested in the brain tissue of experimental animals. Esters of pGlu-Glu-Pro-NH(2) with long-chain primary alcohols emerged as potentially useful prodrugs to improve the central nervous system activity of pGlu-Glu-Pro-NH(2) upon systemic administration, as revealed by the enhancement of analeptic activity in mice.

Neuropharmacodynamic evaluation of the centrally active thyrotropin-releasing hormone analogue [Leu2]TRH and its chemical brain-targeting system.

The centrally active thyrotropin-releasing hormone (TRH) analogue pGlu-Leu-Pro-NH(2) ([Leu(2)]TRH) showed a significant increase in the extracellular acetylcholine concentration during its perfusion to the hippocampus in rats, and this effect was manifested upon the delivery of the analogue in much smaller quantities compared to TRH when measured by in vivo intracranial microdialysis. The neuropharmacodynamic efficacy of [Leu(2)]TRH upon intravenous administration was augmented by the use of a brain-targeting derivative in which the progenitor sequence of the mature peptide was embedded in a molecular architecture that promoted enhanced brain delivery, retention and in situ generation of the pharmacologically active molecule. Compared to the unmodified peptide, the targeting system significantly improved the cumulative effect of the treatment on extracellular acetylcholine levels in rats.

Design, synthesis, and biological evaluation of novel, centrally-acting thyrotropin-releasing hormone analogues.

Novel, metabolically stable and centrally acting TRH analogues with substituted pyridinium moieties replacing the [His(2)] residue of the endogenous peptide were prepared by solid-phase Zincke reaction. The 1,4-dihydropyridine prodrugs of these analogues obtained after reducing the pyridinium moiety were able to reach the brain and maintain a sustained concentration of the charged, degradation-resistant analogues formed after enzymatic oxidation of the prodrug, as manifested by the analeptic action measured in mice. Among the four analogues reported, compound 2a showed the highest potency and longest duration of action in reducing the pentobarbital-induced sleeping time compared to the parent TRH. No binding to the endocrine TRH-receptor was measured for 2a; thus, this compound emerged as a potent, centrally acting TRH analogue.