Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments

Vasopressin is involved in memory processes. A single subcutaneous injection of arginine-8-vasopressin (AVP) increases resistance to extinction of a pole jumping avoidance response. This effect can also be achieved after a single intraventricular administration of much lower amounts than after systemic injection. The covalent ring of AVP, pressinamide (PA), is also highly active following intraventricular administration while the C-terminal part prolyl-arginyl-glycinamide (PAG) is less active. These results indicate that the covalent ring of vasopressin contains the essential requirements for the behavioral effect of this neurohormone. A second activity site however may be present in the C-terminal portion of the molecule.     

Competitive binding of dynorphin-(1-13) and beta-endorphin to cerebroside sulfate in solution

Circular dichroism was used as a probe for competitive binding of two opioid peptides, dynorphin-(1-13) and beta-endorphin, with cerebroside sulfate, a membrane lipid thought to be part of the morphine receptor complex. The rationale was that bound beta-endorphin is partially helical but bound dynorphin-(1-13) remains unordered, thus making it possible to detect the degree of binding of beta-endorphin. The addition of dynorphin-(1-13) to a cerebroside sulfate solution of beta-endorphin invariably displaced beta-endorphin from the peptide-lipid complex, but the addition of beta-endorphin had little effect on dynorphin-(1-13) bound to the lipid. Similar results were obtained for competitive binding of the two peptides with two other amphiphiles, sodium dodecyl and decyl sulfate. The maximum number of binding sites on dynorphin-(1-13) and beta-endorphin was between five and six, which coincides with the five positively charged side chains plus an alpha NH+3 group at the NH2 terminus on both peptide molecules. The results support our working hypothesis that dynorphin-(1-13) may displace beta-endorphin bound to the receptor, which in turn can account for the inhibition of beta-endorphin-induced analgesia by dynorphin-(1-13).     

Intrathecal administration of beta-endorphin and dynorphin-(1-13) for the treatment of intractable pain

Seven cases of chronic pain were treated by intrathecal administration of 30 micrograms of beta-endorphin and dynorphin-(1-13). Compared with saline, both peptides were able to suppress pain for periods up to 4.5 and 7 hours on the average, respectively. No significant side reactions were noticed during the entire investigation.     

Comparative structure-function studies with analogs of dynorphin-(1-13) and [Leu5]enkephalin

Analogs of dynorphin-(1-13) with modifications in the enkephalin segment were compared with correspondingly modified analogs of [Leu5]enkephalin in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in mu- and delta-receptor selective binding assays. The obtained results indicate that a) the enkephalin binding domain of the dynorphin (kappa) receptor has structural requirements which are distinct from those of the enkephalin binding site at the mu-receptor and b) the introduction of an identical conformational constraint in [Leu5]enkephalin and in the enkephalin segment of dynorphin-(1-13) produces a superpotent agonist in both cases. Fluorescence energy transfer measurements with the active [4-tryptophan]analogs of dynorphin-(1-13) and [Leu5]enkephalin and with dynorphin-(1-17) demonstrated a more extended conformation of the N-terminal tetrapeptide segment in [Trp4]dynorphin-(1-13) than in [Trp4, Leu5]enkephalin as well as the absence of an interaction between the N- and C-terminal segments of dynorphin-(1-17).     

Synthesis and biological activity of dynorphin-(1-13) and analogs substituted in positions 8 and 10

Dynorphin-(1-13) (Dyn-(1-13)) and various analogs substituted in positions 8 and 10 were synthesized by the solid-phase technique and analyzed for their ability to inhibit the electrically evoked contraction of the guinea pig ileum (GPI) and to compete with the binding of [3H]-ethylketocyclazocine (EKC, kappa ligand), [3H]-[D-Ala2, MePhe4-Gly-ol5]-enkephalin (DAGO, mu ligand) and [3H]-[D-Ser2, Thr6]-Leu-enkephalin (DSLET, delta ligand) to membrane preparations of the guinea pig cerebellum or rat brain. Introduction of Ala in position 8 decreased the activity of the peptide on the GPI by 50% but induced a 2.22-fold increase in its affinity for the kappa receptor ([3H]-EKC binding displacement from guinea pig cerebellum; Ki of 0.05 nM as compared with 0.11 nM for Dyn-(1-13)). On the other hand, the ability of [Ala8] Dyn-(1-13) to displace the binding of [3H]-DSLET from rat brain membranes was decreased by a factor of 1.7 while its affinity for the mu receptor was not greatly affected ([3H]-DAGO displacement; Ki of 0.44 nM as compared with 0.50 nM for Dyn-(1-13)). Replacement of position 8 by D-Ala caused similar changes in the activity of the peptide but the increase in its affinity for the kappa site was somewhat smaller (Ki of 0.08 nM as compared with 0.11 nM). [D-Pro10]-Dyn-(1-13) was equipotent to [Ala8]-Dyn-(1-13) in the GPI but its affinity for the mu binding site was decreased by a factor of 2.7 as compared with Dyn-(1-13).(ABSTRACT TRUNCATED AT 250 WORDS)     

Blockade effects of (Nphe1)Nociceptin(1-13)-NH(2) on anti-nociception induced by intrathecal administration of nociceptin in rats

The present study investigated the roles of the opioid-receptor-like (ORL1) receptor and its endogenous ligand nociceptin on nociception in the spinal cord of rats. Intrathecal administration of 10 nmol of nociceptin produced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulation. There were no significant changes of average maximum angles in inclined plane tests after intrathecal injection of 10 nmol of nociceptin in rats. The intrathecal nociceptin-induced increases in HWL were antagonized by intrathecal administration of (Nphe1)Nociceptin(1-13)-NH(2), a selective antagonist of ORL1 receptor, in a dose-dependent manner. The results demonstrated that ORL1 receptor is involved in the nociceptin-induced anti-nociceptive effect in the spinal cord of rats.     

Syntheses and reactions of methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate

The syntheses and reactions of two epoxyketoacids (methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (IV) and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (V)) are described. The synthetic method is based on the stereoselective oxidation of linoleic acid by soybean lipoxygenase to produce the corresponding 13-hydroperoxide. Reduction of the hydroperoxide with sodium borohydride followed by oxidation, esterification and epoxidation yielded the compounds IV and V with a global yield of 14% and 3%, respectively, referred to the diasteromerically pure isolated compounds. Confirmation of the structures was carried out by reduction of the ketone group with sodium borohydride and by the opening of the oxirane ring with methanolic boron trifluoride. The reduction of compounds IV and V with hydrogen mainly yielded the tetrahydrofuranoid fatty acid, methyl 10,13-epoxyoctadecanoate. This reaction may be considered a new procedure to obtain tetrahydrofuranoid fatty acids.     

Behavioral and neurochemical effects of the new opioid peptide dynorphin--(1-13): comparison with other neuropeptides

This study was undertaken to determine whether the recently discovered opioid-like peptide dynorphin-(1–13) could influence both excessive grooming in the rat and also the activity of the ACTH-sensitive B-50 protein kinase in vitro. Dynorphin-(1–13), when injected intracerebroventricularly at a dose of 1 to 10 μg, resulted in excessive grooming behavior similar to that observed after administration of ACTH-(1–24). In contrast, leu-enkephalin was not effective in the same dose-range. The grooming behavior elicited by both ACTH-(1–24) and dynorphin-(1–13) was blocked by pre-treatment of the rats with naloxone. Furthermore we observed that dynorphin-(1–13) and ACTH-(1–24) were potent inhibitors of B-50 protein kinase. Leu-enkephalin was not effective whereas β-endorphin was a relatively weak inhibitor. Naloxone did not block these in vitro effects. The relationship of these ohenomena to the opioid receptor is discussed.     

Photophysical properties of Eu(SiW11O39)2 and Eu(BW11O39)2

The photophysical properties (absorption, emission, and excitation spectra; luminescence quantum yields; luminescence decay lifetimes ) of K₁₃[Eu(SiW₁₁O₃₉)₂] and K₁₅[Eu(BW₁₁O₃₉)₂] in aqueous solution and in the solid state are reported. Both complexes exhibit broad and very intense O → W charge transfer bands in the U.V. region and weak and narrow f → f Eu₃₊ bands in the visible. At 77 K the luminescence emission of both complexes, which consists of ⁵D_O → ⁷F_J bands split by the local crystal field, can be pumped very efficiently via both the O → W CT and the f → f Eu³⁺ levels, whereas at 298 K only pumping via the f → f Eu³⁺ is efficient. The values of the luminescence decay lifetimes in H₂O and D₂O solution are quite similar, showing that no water molecule is coordinated to the central Eu₃₊ ion. The high resolution emission spectra are discussed in an attempt to define the coordination symmetry of Eu₃₊.     

Solid phase (9-fluorenylmethyloxycarbonyl) synthesis and characterization of [[1-13C]Phe11]gramicidin B1

The solid phase synthesis of [[1-¹³C]Phe¹¹]Gramicidin B was successfully achieved using the 9-Fluorenylmethyloxycarbonyl protecting group. There was at least a 30% drop in yield during the adition of the three valines in positions 8, 7 and 6, which is likely due to steric hindrance caused by the steric constraints of the valine side chains. Still, the overall yield of the peptide was comparable with that obtained using thetert-butyloxycarbonyl group for protection. The synthetic Gramicidin B was completely characterized by high-pressure liquid chromatography, circular dichroism, and¹³C nuclear magnetic resonance spectra. Also reported are the single-channel conductance properties, which compare favorably with those reported earlier and demonstrate the interesting multiplicity of conductance states to be distinquishable from that of Gramicidin A. Thus, these several useful characterizations have been carried out on the same preparation.Abbreviations: IUPAC-IUB Commission recommendations are used in most cases: AcOH, acetic acid; Boc,tert-butyloxycarbonyl; CD, circular dichroism; CMR,¹³C nuclear magnetic resonance; DCC,N,N'-dicyclohexylcarbodiimide; EtOAc, ethyl acetate; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBt, 1-hydroxybenzot iazole; HPLC, high-pressure liquid chromatography; MeOH, methanol;-OSu,N-hydroxysuccinimide ester; TFA, trifluoroacetic acid; t.l.c., thin-layer chromatography; Me₂SO-d ₆, dimethyl-d ₆-sulfoxide; CMA, chloroform-methanol-acetic acid; DMF, dimethyl for-mamide; DIEA, diisoproprylethylamine.     

Behavioral effects of dynorphin1–13 in the mouse and rat: Initial observations

Dynorphin is a recently identified, pharmacologically potent endogenous opioid peptide. Heretofore it has not been characterized for its behavioral effects. The effects of centrally infused dynorphin upon a variety of behaviors were therefore examined in mice and rats. The present findings point to a specific profile of behavioral activity. The peptide was active in facilitating feeding and grooming, but was inactive in modifying pain sensitivity and rearing behavior. Naloxone was generally ineffective in reversing behavioral effects. Dynorphin thus appears to have some opiate-like effects upon exogenous administration but may be rapidly broken down into a behaviorally potent non-opiate peptide fragment.     

Behavioral effects of dynorphin1–13 in the mouse and rat: Initial observations

Dynorphin is a recently identified, pharmacologically potent endogenous opioid peptide. Heretofore it has not been characterized for its behavioral effects. The effects of centrally infused dynorphin upon a variety of behaviors were therefore examined in mice and rats. The present findings point to a specific profile of behavioral activity. The peptide was active in facilitating feeding and grooming, but was inactive in modifying pain sensitivity and rearing behavior. Naloxone was generally ineffective in reversing behavioral effects. Dynorphin thus appears to have some opiate-like effects upon exogenous administration but may be rapidly broken down into a behaviorally potent non-opiate peptide fragment.     

Enantioselective LC/ESI-MS/MS analysis and pharmacokinetic and tissue distribution study of (2RS)-1-(7-methoxy-1H-indol-4-yloxy)-3-(2-(2-methoxyphenoxy)ethylamino)-propan-2-ol in rats

A sensitive and stereospecific liquid chromatography‐tandem mass spectrometry method for the quantitative determination of TWo8 enantiomers ((2RS)‐1‐(7‐methoxy‐1H‐indol‐4‐yloxy)‐3‐(2‐(2‐methoxyphenoxy)ethylamino)‐propan‐2‐ol) was developed and validated in rat serum and some tissues. Racemic TWo8 is a new chemical entity, and it has been shown to possess pharmacological activity in vivo. The assay involved the diastereomeric derivatization of racemic TWo8 with 2,3,4,6‐tetra‐O‐acetyl‐beta‐glucopyranosyl isothiocyanate. The TWo8 diastereoisomers quantification was performed on a triple quadrupole mass spectrometer employing an electrospray ionization technique. The precursor to the product ion transition for TWo8 derivatives and for the internal standard (carbamazepine) was m/z 776.4 → 387.2 and 237.4 → 194.4, respectively. The assay was validated with a linear range of 10–2000 ng/ml of racemic TWo8. The inter‐day precisions for (?)‐(S)‐TWo8 and (+)‐(R)‐TWo8 were 2.1% to 14.9% and 1.3% to 14.8%, respectively. The inter‐day accuracy for (?)‐(S)‐TWo8 and (+)‐(R)‐TWo8 was within 86% to 114% and 91% to 114%, respectively. A pilot pharmacokinetic study of this new β‐adrenolytic compound has shown that (?)‐(S)‐TWo8 is eliminated faster than its antipode. The terminal half‐lives of (?)‐(S)‐TWo8 and (+)‐(R)‐TWo8 were 3.2 and 3.9 h, respectively. The compound distribution into different organs, evaluated in tissue homogenate samples following TWo8 intravenous administration, showed an enantioselective penetration of TWo8 enantiomers in the liver (p < 0.03), in the kidney (p < 0.001), and in the lungs (p < 0.05). The developed method using liquid chromatography‐tandem mass spectrometry method with electrospray ionization could be employed for quantitative determination of compounds with similar structure. Chirality 24:591–599, 2012. © 2012 Wiley Periodicals, Inc.     

Bimodal effects of chronically administered neurokinin B (NKB) on in vivo and in vitro cardiovascular responses in female rats

The in vivo cardiovascular effects of acutely administered neurokinin B (NKB) have been attributed both to direct effects on vascular tone and to indirect effects on central neuroendocrine control of the circulation. We proposed: 1) that a modest long-term increase in plasma NKB levels would decrease mean arterial pressure (MAP) due to attenuated peripheral vascular tone, and 2) that chronic high-dose NKB would increase MAP, due to increased sympathetic outflow which would override the peripheral vasodilation. We examined the in vivo and in vitro cardiovascular effects of chronic peripheral NKB. Low- (1.8 nmol/h) or high- (20 nmol/h) dose NKB was infused into conscious female rats bearing telemetric pressure transducers. MAP, heart rate (HR) and the pressor responses to I.V. phenylephrine (PE, 8 microg) and angiotensin II (Ang II, 150 ng) were measured. Concentration-response curves of small mesenteric arteries were constructed to PE using wire myography. Low-dose NKB reduced basal MAP (88+/-2 mm Hg to 83+/-2 mm Hg), did not affect resting HR, reduced the pressor responses to PE, and attenuated the maximal constriction of mesenteric arteries to PE and KCl. By contrast, high-dose NKB increased basal MAP (86+/-1 mm Hg to 89+/-1 mm Hg), increased HR (350+/-3 beats/min to 371+/-3 beats/min), increased the pressor responses to Ang II and, contrary to our hypothesis, increased the maximum contractile responses of mesenteric arteries to PE and KCl. The cardiovascular effects of NKB are thus dose-dependent: whereas chronic low-dose NKB directly modulates vascular tone to reduce blood pressure, chronic high-dose NKB induces an increase in blood pressure through both central (indirect) and peripheral (direct) pathways.     

Study in vitro and in vivo of nociceptin/orphanin FQ(1-13)NH2 analogues substituting N-Me-Gly for Gly2 or Gly3

In the present study, two analogues containing N-Me-Gly (Sarcosine, Sar) were synthesized to further investigate the structural-activity relationships of orphanin FQ/nociceptin (OFQ/NC, NC). The replacement of Gly(2) or Gly(3) with Sar increased the flexibility and decreased the hydrophobicity of the N-terminal tetrapeptide. The activity of the analogues was investigated in a series of assays in vivo and in vitro. [Sar(2)]NC(1-13)NH(2) was found to (1) produce dose-dependent inhibition of the electrically induced contraction in MVD assay (pEC(50) = 6.14); (2) produce significant hyperalgesia effects in a dose-dependent manner when intracerebroventricularly (i.c.v.) injected in mice. The inhibitive effects of [Sar(2)]NC(1-13)NH(2) in MVD assay could be significantly antagonized by [Nphe(1)]NC(1-13)NH(2), and partially antagonized by naloxone; the hyperalgesic effect of [Sar(2)]NC(1-13)NH(2) could be significantly antagonized by naloxone, and partially antagonized by [Nphe(1)]NC(1-13)NH(2). On the contrary, [Sar(3)]NC(1-13)NH(2) showed no effects in these assays. All the findings suggest that the flexibility of the peptide bond between Phe(1) and Gly(2) and between Gly(2) and Gly(3) play an important role in NC-OP(4) receptor interaction, and the hydrophobicity of the N-terminal tetrapeptide showed no significant effect on this interaction. The present work also helps to provide a novel method to elucidate structural and conformational requirements of the opioid peptide-receptor interaction.     

A quantitative analysis of the shaking behavior induced in rats by β-endorphin and [D-Ala2, Met5] enkephalinamide

β-Endorphin (5–80 μg) or [D-Ala², Met⁵] enkephalinamide (DALA) (5–40 μg) was administered intracerebroventricularly to rats. With both opioid peptides, there was no direct relationship between log dose and mean number of wet-dog shakes (WDS) that occured during the following 15 min. When the results were analyzed quantally, the dose of DALA that caused 50% of the rats to shake at least twice was 8.6 μg (4.9–15 μg). β-Endorphin had such poor efficacy that an ED 50 could not be obtained. Morphine (1 and 5 mg/kg, s.c.) antagonized shaking caused by the optimal dose of DALA (20 μg). Naloxone (0.1–10 mg/kg, s.c.) attenuated both DALA- and β-endorphin-induced WDS in a dose-related manner. This latter result differentiates shaking associated with opioid peptides from that caused by thyrotropin releasing hormone (TRH), another endogenous stimulant of WDS in rats. There was no cross-tolerance between RX 336-M (7,8-dihydro-5′,6′-dimethylylohex-5′-eno-1′,2′,8′,14 codeinone), a novel shake inducing agent, and β-endorphin. This finding again differentiates β-endorphin-induced shaking from that caused by TRH and also from that associated with several exogenous stimulants of WDS.     

2-(aminomethyl)-2-decarboxyprostaglandin F2α type analogs

The 2-(aminomethyl)-2-decarboxy analogs of prostaglandin F₂α (PGF₂α), (15S)-15-methyl-PGF₂α, 16-phenoxy-ω-tetranor-PGF₂α and 16,16-dimethyl-PGF₂α were synthesized. The amino analogs closely resemble the parent PGF₂α compounds as antifertility agents in the hamster.