Oxygen as an important factor modulating in vitro MeHgCl toxicity associated with mitochondrial genes in hiPSCs.

Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.

Nitric oxide as a modulator in platelet- and endothelium-dependent antithrombotic effect of eplerenone in diabetic rats.

We have recently demonstrated the antithrombotic effect of eplerenone on the arterial thrombotic process in diabetic rats associated with suppression of coagulation and enhancement of fibrinolysis. The aim of this study was to evaluate the role of platelets and endothelium in the mechanism of eplerenone antithrombotic action. Diabetes was induced in male Wistar rats with a single injection of streptozotocin (65 mg/kg). On the 25th day, treatment with eplerenone (100 mg/kg) was initiated for 10 days. Eplerenone did not change hemodynamic parameters (blood pressure, carotid blood flow, and heart rate), however, improved endothelium-dependent vasorelaxation in aortas and small mesenteric arteries, enhanced the aortic amounts of mRNA of endothelial nitric oxide synthase (eNOS), and reduced mRNA of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 2. A prolongation of bleeding time and decrease in platelet adhesion to collagen ex vivo was also observed. These changes were accompanied by prolonged time to occlusion and increased blood flow, and finally reduced thrombus mass in diabetic rats. The inhibition of NOS with L-NAME reduced the eplerenone antithrombotic effect. Our study provides evidence that the antithrombotic effect of eplerenone in diabetic rats is nitric oxide-dependent and associated with inhibiting the adhesion of platelets, as well as normalizing endothelial function. The mechanism of eplerenone antithrombotic action in diabetes is a result of improved endothelial nitric oxide bioavailability that leads to the improvement vascular and platelet function.

Developmental stage dependent neural stem cells sensitivity to methylmercury chloride on different biofunctional surfaces.

Sensitivity of neural stem cells viability, proliferation and differentiation upon exposure to methylmercury chloride (MeHgCl) was investigated on different types of biofunctional surfaces. Patterns of biodomains created by microprinting/microspotting of poly-l-lysine or extracellular matrix proteins (fibronectin and vitronectin) allowed for non-specific electrostatic or specific, receptor mediated interactions, respectively, between stem cells and the surface. The neural stem cell line HUCB-NSC has been previously shown to be susceptible to MeHgCl in developmentally dependent manner. Here we demonstrated that developmental sensitivity of HUCB-NSC to MeHgCl depends upon the type of adhesive biomolecules and the geometry of biodomains. Proliferation of HUCB-NSC was diminished in time and MeHgCl concentration dependent manner. In addition, the response to MeHgCl was found to be cell-type dependent. Undifferentiated cells were the most sensitive independently of the type of bioactive domain. Significant decrease of GFAP+ cells was detected among cells growing on poly-l-lysine, while on fibronectin and vitronectin, this effect was observed only in the highest (1µM) concentration of MeHgCl. ß-Tubulin III expressing cells were most sensitive on fibronectin domains. In addition, limited bioactive domains to µm in size, as compared to non-patterned larger area of the same adhesive substrate, exerted protective role. Thus, the surface area and type of cell/biofunctional surface interaction exerted significant influence on developmental stage and cell-type specific response of HUCB-NSC to MeHgCl.

The relationship between the dimensions of the right coronary artery and the type of coronary vasculature in human foetuses.

BACKGROUND: The area of vascular supply of particular coronary arteries is directly linked to the varying typology of the coronary vasculature. This factor may have a significant influence on the coronary vessel diameters. To date there has been no published research that analyses the relationship between the type of coronary vasculature and the dimensions of the epicardial arteries in the human foetus. There are only a few papers that deal with this issue in the postnatal period of human life. MATERIAL AND METHODS: The study was carried out on a group of 187 human foetuses aged five to seven months of intrauterine life. Prior to examination all foetuses had been conserved in a 9% formaldehyde solution for a minimum of three months. All foetuses had been aborted naturally. None of them had any external signs of malformations or developmental abnormalities. The number of foetuses in the particular age groups was variable. Adachi/Bianchi classification was used to categorize the particular vasculature types: type I -- classic, neither artery is dominating; type II -- dominant right coronary artery; type III -- dominant left coronary artery. RESULTS AND CONCLUSIONS: The analysis of differences between the artery dimensions in particular types of coronary vasculature revealed that such differences existed between types I and II and also between types II and III.

Four close bupranolol analogues are antagonists at the low-affinity state of beta1-adrenoceptors.

Bupranolol is an antagonist at the cardiostimulatory low-affinity state of b(1)-adrenoceptors and we were interested whether this effect is shared by its fluorine (GD-6), methyl (DZ-51) and isopropyl analogue (DZ-13) and by the analogue hydroxylated at the tertiary butyl moiety (DZ-52). (-)-Bupranolol and compounds (-)-GD-6, (+)-GD-6, (-)-DZ-13, (+)-DZ-13, DZ-51 and DZ-52 antagonized the CGP 12177-induced tachycardia in pithed rats with "apparent pA(2) values" of 6.1, 6.1, 4.6, 5.5, 4.6, 5.1 and 5.3, respectively. For comparison, their potencies and affinities at the high-affinity state of b(1)-adrenoceptors were determined, using the xamoterol-induced tachycardia in pithed rats and [(3)H]CGP 12177 binding to rat cerebrocortical membranes. The respective "apparent pA(2) values" in the functional experiments were 7.9, 8.1, 5.4, 8.4, 5.7, 7.3 and 6.8 and the pK(i) values in the binding experiments were 8.8, 8.4, 6.9, 8.5, 6.7, 8.4 and 8.2. In conclusion, (-)-bupranolol and its fluorine analogue (-)-GD-6 are equipotent at the low-affinity state of beta(1)-adrenoceptors. The stereoselectivity of GD-6 and DZ-13 suggests that the low-affinity state is indeed a receptor.

Influence of fermentation conditions on glucosinolates, ascorbigen, and ascorbic acid content in white cabbage (Brassica oleracea var. capitata cv. Taler) cultivated in different seasons.

The content of glucosinolates (GLS), ascorbigen, and ascorbic acid in white cabbage (Brassica oleracea var. capitata cv. Taler) cultivated in different seasons (summer and winter) was determined, before and after spontaneous and starter-induced fermentation. Different salt concentrations (0.5% NaCl or 1.5% NaCl) were used for sauerkraut production. Glucoiberin, sinigrin, and glucobrassicin were dominating in raw white cabbage cultivated either in winter or summer seasons. Ascorbigen precursor, glucobrassicin, was found higher in cabbage cultivated in winter (2.54 micromol/g dw) than those grown in summer (1.83 micromol/g dw). Cabbage fermented for 7 d was found to contain only traces of some GLS irrespective of the fermentation conditions used. Ascorbigen synthesis occurred during white cabbage fermentation. Brining cabbage at low salt concentration (0.5% NaCl) improved ascorbigen content in sauerkraut after 7 d of fermentation at 25 degrees C. The highest ascorbigen concentration was observed in low-sodium (0.5% NaCl) sauerkraut produced from cabbage cultivated in winter submitted to either natural (109.0 micromol/100 g dw) or starter-induced fermentation (108.3 and 104.6 micromol/100 g dw in cabbages fermented by L. plantarum and L. mesenteroides, respectively). Ascorbic acid content was found higher in cabbage cultivated in summer and fermentation process led to significant reductions. Therefore, the selection of cabbages with high glucobrassicin content and the production of low-sodium sauerkrauts may provide enhanced health benefits towards prevention of chronic diseases.

Virodhamine relaxes the human pulmonary artery through the endothelial cannabinoid receptor and indirectly through a COX product.

BACKGROUND AND PURPOSE: The endocannabinoid virodhamine is a partial agonist at the cannabinoid CB(1) receptor and a full agonist at the CB(2) receptor, and relaxes rat mesenteric arteries through endothelial cannabinoid receptors. Its concentration in the periphery exceeds that of the endocannabinoid anandamide. Here, we examined the influence of virodhamine on the human pulmonary artery. EXPERIMENTAL APPROACH: Isolated human pulmonary arteries were obtained during resections for lung carcinoma. Vasorelaxant effects of virodhamine were examined on endothelium-intact vessels precontracted with 5-HT or KCl. KEY RESULTS: Virodhamine, unlike WIN 55,212-2, relaxed 5-HT-precontracted vessels concentration dependently. The effect of virodhamine was reduced by endothelium denudation, two antagonists of the endothelial cannabinoid receptor, cannabidiol and O-1918, and a high concentration of the CB(1) receptor antagonist rimonabant (5 muM), but only slightly attenuated by the NOS inhibitor L-NAME and not affected by a lower concentration of rimonabant (100 nM) or by the CB(2) and vanilloid receptor antagonists SR 144528 and capsazepine, respectively. The COX inhibitor indomethacin and the fatty acid amide hydrolase inhibitor URB597 and combined administration of selective blockers of small (apamin) and intermediate and large (charybdotoxin) conductance Ca(2+)-activated K(+) channels attenuated virodhamine-induced relaxation. The vasorelaxant potency of virodhamine was lower in KCl- than in 5-HT-precontracted preparations. CONCLUSIONS AND IMPLICATIONS: Virodhamine relaxes the human pulmonary artery through the putative endothelial cannabinoid receptor and indirectly through a COX-derived vasorelaxant prostanoid formed from the virodhamine metabolite, arachidonic acid. One or both of these mechanisms may stimulate vasorelaxant Ca(2+)-activated K(+) channels.

Potential involvement of a propranolol-insensitive atypical beta-adrenoceptor the vasodilator effect of cyanopindolol in the human pulmonary artery.

The aim of our study was to examine whether non beta(1)-/beta(2)-adrenoceptors participate in the relaxation of the human pulmonary artery. For this purpose the vasodilatory effect of the non-conventional partial beta-adrenoceptor agonist cyanopindolol was examined. Cyanopindolol (1-300 microM), studied in the presence of the beta(1)-/beta(2)-adrenoceptor antagonist propranolol, relaxed the human pulmonary artery preconstricted with serotonin 1 microM in a concentration-dependent manner (maximally by about 80%). This effect was diminished by bupranolol 10 microM (an antagonist of beta(1)-beta(3)-adrenoceptors and the low affinity state of the beta(1)-adrenoceptor) and CGP 20712 10 microM (known to antagonize the low-affinity state of the beta(1)-adrenoceptor at high concentrations). In further experiments, the effect of beta-adrenoceptor ligands on the serotonin-induced vasoconstriction was examined. The concentration-response curve for serotonin was not affected by cyanopindolol 30 microM, bupranolol 10 microM and CGP 20712 10 microM but shifted to the right by cyanopindolol 100 and 300 microM; the serotonin 5-HT(2A) receptor antagonist ketanserin 0.3 microM abolished the maximum contraction elicited by serotonin. In conclusion, the present study reveals that the vasodilatory effect of cyanopindolol in the human pulmonary artery consists of two components, i.e. activation of a propranolol-insensitive atypical beta-adrenoceptor and antagonism against 5-HT(2A) receptors.

Neurogenic potential of human umbilical cord blood: neural-like stem cells depend on previous long-term culture conditions.

In vitro studies conducted by our research group documented that neural progenitor cells can be selected from human umbilical cord blood (HUCB-NPs). Due to further expansion of these cells we have established the first human umbilical cord blood-derived neural-like stem cell line (HUCB-NSC) growing in serum-free (SF) or low-serum (LS) medium for over 3 years. The purpose of the study was to evaluate the neurogenic potential of HUCB-NSCs cultured in SF and LS condition in different in vitro settings before transplantation. We have shown that the number of cells attaining neuronal features was significantly higher for cultures expanded in LS than in SF condition. Moreover, the presence of neuromorphogens, cultured rat astrocytes or hippocampal slices promoted further differentiation of HUCB-NSCs into neural lineage much more effectively when the cells had derived from LS cultures. The highest response was observed in the case of co-cultures with rat primary astrocytes as well as hippocampal organotypic slices. However, the LS cells co-cultured with hippocampal slices expressed exclusively a set of early and late neuronal markers whereas no detection of cells with glial-specific markers was possible. In conclusion, certain level of stem/progenitor cell commitment is important for optimal response of HUCB-NSC on the neurogenic signals provided by surrounding environment in vitro.

Mercury distribution in the skin of beluga (Delphinapterus leucas) and narwhal (Monodon monoceros) from the Canadian Arctic and mercury burdens and excretion by moulting.

Beluga and narwhal skin as a whole (in Inuktitut known as "muktuk") is considered to be a delicacy by native Canadian and Greenland people. Individual strata of the skin, and muscle from 27 beluga from the western, and 20 narwhal from the eastern Canadian Arctic, were analyzed for mercury and the thickness and density of each skin layer was measured. Mercury was not uniformly distributed in the skin, but increased outwardly with each layer. The concentration was only 0.29 and 0.16 microg/g (wet wt) in the innermost layer (dermis) of belugas and narwhal respectively, and 1.5 and 1.4 microg/g (wet wt) in the outermost layer (degenerative epidermis) of beluga and narwhal, respectively. There was a significant (alpha=0.05) association between age and mercury concentration in each skin layer, the regression coefficients progressively increasing from the inner layer (dermis) to the outer layer: 0.011-0.063 microg/g year-1; 0.034 microg/g year-1 for skin as a whole; 0.054 microg/g year-1 for muscle. The concentration of total mercury was 0.84 and 0.59 microg/g (wet wt) in skin as a whole (muktuk) of beluga and narwhal respectively, and 0.12 and 0.03 microg/g in blubber, respectively. The average, total mercury concentration in muscle tissue was 1.4 and 0.81 microg/g wet wt, in beluga and narwhal respectively, exceeding (except for blubber) the Canadian Government's Guideline (0.5 microg/g wet wt) for fish export and consumption. The skin surface area of an average-size beluga and narwhal was estimated (6.10 and 6.50 m2, respectively), as were excretions of mercury through moulting (13,861 and 6721 microg year-1; 14 and 7 mg year-1) for belugas and narwhal, respectively. The whole-body mercury burden (699,300 microg; 700 mg) for a 1000 kg beluga and its various tissues were estimated, as was the fraction of mercury excreted by moulting (2-0.42% of the whole-body burden). Annual mercury burden increments in beluga skin, muscle and the whole body were estimated (2750; 17,280; 40,00 microg year-1, respectively), using regression coefficients of age on mercury concentration. The annual gross mercury intake via food was estimated (131,400 microg), of which 70% was excreted.

Novel histaprodifen analogues as potent histamine H1-receptor agonists in the pithed and in the anaesthetized rat.

We have shown previously that histaprodifen and its Nalpha-substituted analogues methylhistaprodifen and dimethylhistaprodifen are highly potent H1-receptor agonists in vivo. The aim of the present study was to examine the influence of four newly synthesized histaprodifen analogues, 3-fluoro-methylhistaprodifen (1), Nalpha-imidazolylethylhistaprodifen (2), bis-histaprodifen (3) and Nalpha-methyl-bis-histaprodifen (4), on the cardiovascular system in the pithed and in the anaesthetized rat. In pithed and vagotomized rats, diastolic blood pressure (which was increased to 80-85 mmHg by vasopressin infusion) was decreased dose dependently by methylhistaprodifen (the reference compound) and by compounds 1-4. The maximum decrease was about 47-50 mmHg for methylhistaprodifen and compounds 1, 2 and 3. Their potencies, expressed as pED50 (the negative logarithm of the dose in mole per kilogram body weight that decreased diastolic blood pressure by 25 mmHg), were 8.31, 8.23, 8.26 and 7.84, respectively. With compound 4 the maximal effect was not achieved at doses up to 1 micromol/kg (the latter dose decreased blood pressure by about 30 mmHg; pED50 approximately 6.5). The vasodepressor effect of the five compounds was attenuated by the H1-receptor antagonist dimetindene (1 micromol/kg) but was not changed by combined administration of the H2- and H3-receptor antagonists ranitidine and thioperamide (1 micromol/kg each), by combined administration of the alpha1- and alpha2-adrenoceptor antagonists prazosin and rauwolscine (1 micromol/kg each) or by the beta-adrenoceptor antagonist propranolol (3 micromol/kg). In anaesthetized rats methylhistaprodifen and compounds 1-4 induced almost the same fall in blood pressure as in pithed and vagotomized animals; the effects were sensitive to blockade by dimetindene (1 micromol/kg). Higher doses of compounds 1 and 2 (1 micromol/kg) increased heart rate in pithed and vagotomized rats in a manner sensitive to propranolol (3 micromol/kg) but insensitive to dimetindene (3 micromol/kg). The same dose of methylhistaprodifen and of compounds 3 and 4 failed to affect heart rate. We conclude that the agonistic potency of compounds 1 and 2 at H1-receptors in the cardiovascular system of the rat equals that of methylhistaprodifen, the most potent histamine H1-receptor agonist available until recently. Compounds 1 and 2 exhibit sympathomimetic activity at high doses.

Toxicity and toxicokinetics of a phosphorothioate oligonucleotide against the c-myc oncogene in cynomolgus monkeys.

A 2-week toxicity and toxicokinetic study of a 15-mer phosphorothioate oligonucleotide, INX-3280, against the c-myc oncogene was performed in cynomolgus monkeys. As this oligonucleotide readily adopts an aggregate structure, a quadruplex, which may be associated with adverse physiologic effects, this study was performed using INX-3280 that had been converted to its monomeric form. Animals received intravenous (i.v.) infusions of monomeric INX-3280 three times per week for 2 weeks at doses of 3 or 15 mg/kg per administration. The monkeys were examined for clinical signs: changes in hematology, serum chemistry, coagulation, and urinalysis parameters; complement activation; macroscopic findings at necropsy; and histopathologic alterations. In addition, the toxicokinetics of INX-3280 were evaluated, using a validated HPLC assay, after the first and last (sixth) doses. No treatment-related clinical signs of any adverse effects were observed, and there were no test article-related changes in hematology, serum chemistry, or complement activation parameters. The only alteration in clinical pathology parameters was a minor (30%) prolongation of the activated partial thromboplastin time (aPTT), reflecting slight inhibition of the intrinsic coagulation pathway, which was less than that reported with other oligonucleotides given at similar doses. Treatment-related histopathologic alterations consisted of characteristic accumulation of basophilic material in the cytoplasm of tubular epithelial cells in the kidney, resident macrophages in the lymph nodes, and Kupffer cells in the liver. These changes were graded as minimal in all cases. The basophilic material is believed to reflect accumulation of the oligonucleotide or metabolites or both. The pharmacokinetic parameters of INX-3280 were identical on the first and sixth administrations and were similar to those reported for other phosphorothioate oligonucleotides. Maximum concentration (Cmax) values for INX-3280 (101-119 microg/ml) were in excess of the threshold plasma concentrations reported to trigger complement activation by phosphorothioate oligonucleotides. It is concluded that the safety profile of monomeric INX-3280 in cynomolgus monkeys is quite favorable relative to the known effects of other phosphorothioate oligonucleotides, particularly with respect to the blood level-related toxicities of this class of compounds, including complement activation and inhibition of coagulation. This study found no toxicities that were expected to be clinically significant.

Muscular dystrophy in mice caused by two different alterations of dystrophin gene.

The study addressed the question of the functional significance of various isoforms of dystrophin. Mice bearing two different alterations of the dystrophin gene, mdx and mdx-beta geo, were examined. Dystrophic mice with mdx mutation do not express full-length dystrophins, while they express short dystrophins; on the other hand, the dystrophic mice with mdx-beta geo mutation express neither full-length nor short dystrophins. We found pathological changes typical for muscular dystrophy in the diaphragm of both mutant strains. No pathological changes were found in brains of either mdx or mdx-beta geo mutants. We concluded tentatively that short isoforms of dystrophin do not contribute to the muscular dystrophy and that they do not play any role in the development and maintenance of histological structure of the brain.

Content of glucosinolates in cruciferous vegetables grown at the same site for two years under different climatic conditions.

Individual glucosinolates (GLS) were determined in vegetables of three Cruciferae species: Brassica oleracea L. (white cabbage, red cabbage, Savoy cabbage, Brussels sprouts, cauliflower, kale, kohlrabi), Brassica rapa L. (turnip), and Raphanus sativus L. (red radish, black radish, and white radish) produced in two years. The cultivars were compared for the contents of total-, indole-, and aliphatic GLS. In both years, the total content of GLS was highest in black radish, and all examined R. sativus vegetables contained the greatest amount of aliphatic GLS. Neither the level nor the identity of GLS differentiated among the vegetables of the other cultivars grown in the same year. Comparison of the GLS contents of the same cultivar in two production years, which differed in temperature and rainfall rate, showed that low average 10-day rainfall and high average temperature during the vegetation period significantly increased the GLS content of vegetables. This suggests that the year x cultivar interaction modified the GLS content of vegetables.

Antioxidant activity and total phenolics in selected cereal grains and their different morphological fractions.

The purpose of this study was to examine the antioxidant properties of water and 80% methanolic extracts of cereal grains and their different morphological fractions. Wheat (Triticum aestivum L.) cv. Almari and cv. Henika, barley (Hordeum vulgare L.) cv. Gregor and cv. Mobek, rye (Secale cereale L.) cv. Dankowskie Zlote, oat (Avena sativa L.) cv. Slawko and buckwheat (Fagopyrum esculentum Moench) cv. Kora were used. PC (L-alpha-phosphatidylcholine) liposome system and spectrophotometric assay of total antioxidant activity (TAA) were used to evaluate the antioxidative activity of extracts. Among the water extracts, only the one prepared from buckwheat exhibited antioxidant activity at the concentration analyzed. The following hierarchy of antioxidant activity was provided for 80% methanolic extracts originated from whole grain: buckwheat > barley > oat > wheat congruent with rye. The antioxidant activity was observed in extract prepared from separated parts of buckwheat and barley. In respect to hulls, the antioxidant hierarchy was as follows: buckwheat > oat > barley. The correlation coefficient between total phenolic compounds and total antioxidative activity of the extracts was -0.35 for water extracts and 0.96, 0.99, 0.80, and 0.99 for 80% methanolic extracts originated from whole grains, hulls, pericarb with testa fractions and endosperm with embryo fractions, respectively.

Nociceptin inhibits the neurogenic vasopressor response in the pithed rat via prejunctional ORL1 receptors.

Nociceptin (or orphanin FQ), the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, decreases blood pressure in the conscious and anesthetized rat. This study examined whether prejunctional inhibitory ORL1 receptors located on the postganglionic sympathetic neurons innervating the resistance vessels are detectable in pithed rats. In pithed and vagotomized rats electrical stimulation of the preganglionic sympathetic nerve fibers, injection of nicotine (2 micromol/kg) or noradrenaline (1 nmol/kg) increased blood pressure by about 30 mmHg. The electrically induced vasopressor response was decreased dose-dependently by nociceptin (0.001-1 micromol/kg; decrease by about 60% at 1 micromol/kg). Nociceptin 0.1 micromol/kg reduced the nicotine-induced vasopressor response by about 40% but at doses up to 1 micromol/kg failed to affect the increase in blood pressure evoked by noradrenaline. The inhibitory action of nociceptin on the electrically and nicotine-induced increase in blood pressure was attenuated by the ORL1 receptor antagonists naloxone benzoylhydrazone (5 micromol/kg) and/or [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)NH2 (1 micromol/kg) but was not affected by naloxone 10 micromol/kg. In conclusion, the present data suggest that the postganglionic sympathetic nerve fibers innervating the resistance vessels of the rat are endowed with prejunctional ORL1 receptors, activation of which causes inhibition of noradrenaline release.

Acetyl-RYYRIK-NH(2) is a highly efficacious OP(4) receptor agonist in the cardiovascular system of anesthetized rats.

Nociceptin, the endogenous ligand of the OP(4) or ORL(1) (opioid receptor-like(1)) receptor, decreases blood pressure and heart rate in anesthetized rats. Since the OP(4) receptor antagonist [Phe(1)Psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2) possesses an agonistic effect in this model, we examined whether other purported OP(4) receptor antagonists, acetyl-RYYRIK-NH(2) and naloxone benzoylhydrazone, antagonize the depressant effects of nociceptin. Acetyl-RYYRIK-NH(2), like nociceptin and [Phe(1)Psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2) and unlike naloxone benzoylhydrazone, decreased diastolic blood pressure and heart rate (rank order of potencies: nociceptin approximately equal to acetyl-RYYRIK-NH(2) >> [Phe(1)Psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2)). The depressant effects were insensitive to the OP(1-3) receptor antagonist naloxone but diminished by naloxone benzoylhydrazone. In conclusion, the hypotensive and bradycardic effects of nociceptin in the anesthetized rat are mediated via OP(4) receptors, at which acetyl-RYYRIK-NH(2) is a highly potent and efficacious agonist.

A comparative CD and fluorescence study of a series of model calcium-binding peptides.

Lanthanide-saturated peptides analogous to calcium-binding loops of EF-hand proteins can be used to stabilize the alpha-helical structure of peptide or protein segments attached to their C-termini. To study conformational properties of such loop-containing hybrids it is necessary to produce them in bacteria. In peptides obtained in this way the helix will be destabilized by the negatively charged C-terminal alpha-carboxyl groups. We propose to block them by the homoserine lactone. The results presented in this paper indicate that the presence of the lactone even at the C-terminus of the loop does not have any negative effect on the loop helix-nucleation ability. On the other hand, the presence of the alpha-NH3+ at the loop N-terminus leads to a drop of metal-binding constant and loss of the rigid structure of the alpha-helical segment of the loop. The alpha-amino group separated by one glycine residue from the loop N-terminus should also be avoided because it perturbs the conformation of the N-terminal part of the loop and may reduce the loop affinity to lanthanide ions.