Primary sclerosing cholangitis: evaluation with MR cholangiography-a case-control study.

PURPOSE: To determine the accuracy of magnetic resonance (MR) cholangiography for detection of primary sclerosing cholangitis (PSC) and localization of PSC in the biliary tract. MATERIALS AND METHODS: In a prospective case-control study involving 102 patients, the MR cholangiograms obtained in 34 patients with PSC established with endoscopic retrograde cholangiopancreatography (ERCP) were compared with the MR cholangiograms obtained in 68 age-matched control patients with hepatobiliary diseases other than PSC. Two abdominal radiologists conducted an independent, blinded random review of the MR cholangiograms to assess for the presence or absence of PSC and determine the location of PSC in the biliary tract, and then compared the findings with those at ERCP. RESULTS: MR cholangiography was found to be accurate in detecting PSC and in defining the extent of disease. In the detection of PSC, the sensitivities were 88% and 85%; specificities, 97% and 92%; positive predictive values, 94% and 85%; and negative predictive values, 94% and 93% for readers 1 and 2, respectively. Interobserver agreement was excellent (kappa = 0.79). In the localization of extrahepatic PSC, the sensitivities were 83% and 89%; and specificities, 83% and 83% for readers 1 and 2, respectively. Interobserver agreement was good (kappa = 0.62). In the localization of intrahepatic PSC, the sensitivity was 87% for both readers; interobserver agreement was good (kappa = 0.71). CONCLUSION: MR cholangiography enables accurate detection and localization of PSC.

Influence of methylation of the histidine ring of [Sar1]angiotensin II on conformation and biological activity.

[Sar1His(1-Me)6]ANG II and [Sar1His(3-Me)6]ANG II were synthesized by the solid-phase method and purified by reversed-phase HPLC. 1H-NMR spectroscopy at 400 MHz demonstrated the presence of two major conformers for both peptides in DMSO, representing cis and trans isomers (ratio 1:3 and 1:4, respectively) due to restricted rotation at the His-Pro bond. The contractile activities of these peptides in the rat isolated uterus assay were < 0.1 and 27% of that of ANG II, respectively. The bioactivities of these analogues were mirrored in their NMR spectra: the inactive analogue [Sar1His(1-Me)6]ANG II showed perturbations of the Sar and His residues which were not present for [Sar1His(3-Me)6]ANG II and the reference agonist [Sar1]ANG II. The high activity of the analogue methylated at His N3 suggests that an ionizable imidazole proton is not an absolute requirement for expression of biological activity by angiotensin ligands, and that the imidazole group in the molecule may function in an ion dipole-based mechanism when an intramolecular proton transfer (charge relay) mechanism is not available. The biological activity of the ligand appears to depend on the degree of proton transfer from Tyr-OH to the imidazole acceptor, wherein complete (formal) proton transfer represents 100% activity.

Encapsulated liquid crystals as probes for remote thermometry.

A temperature probe based on the magnetic resonance properties of an encapsulated liquid crystal has been investigated. Large changes in magnetic resonance signals occur as the liquid crystal undergoes a phase transition from an anisotropic (nematic) state to the isotropic liquid. The low latent heat of such phase transitions allows for rapid phase changes during a hyperthermia treatment. Transition temperatures can be tailored by adding suitable compounds such as analogues of the liquid crystal or various solvents. Encapsulation is required to maintain the integrity of the liquid crystal, particularly for applications in vivo. Results of preliminary studies designed to demonstrate the technical feasibility of the concept are presented.

Importance of the N-terminal domain of the type I angiotension II antagonist [Sar1,Ile8]ANG II for receptor blockade.

Analogues of the Type I angiotensin (ANG) antagonist, [Sar1,Ile8]ANG II, in which the N-terminal dipeptide was modified were synthesized by the solid phase method and purified by reversed-phase HPLC. Antagonist potencies (pA2) of the peptides were determined on the rat isolated uterus using ANG II as the agonist. Substitution of the Arg residue occupying position 2 of [Sar1,Ile8]ANG II (pA2 8.1) by Gly, Ala, Nle, Phe, Pro or Sar reduced the antagonist potency to pA2 = 7.0, 6.8, 6.7, 6.8, 5.8 and 5.3, respectively. Deletion of the N-terminal Sar residue in these same peptides gave pA2 = 6.8, 5.7, 5.5, 5.9, 6.1 and 7.5, respectively. The characteristically long duration of action of [Sar1,Ile8] was absent for all of these analogues including (des1, Sar2, Ile8]ANG II. These findings demonstrate that the antagonist potencies of Type I angiotensin antagonists for smooth muscle receptors, and also the long duration of action, are dependent on the location of positive charges within the peptide and on the conformation of the molecule in determining favorable electrostatic interactions with the receptor. A model is proposed in which the two positively charged loci on the angiotensin molecule (N-terminus and Arg) interact with two corresponding anionic binding sites on the smooth muscle receptor. The possibility that the prolonged duration of action of [Sar1, Ile8]ANG II results from binding to a different site on the angiotensin receptor from that occupied by ANG II is discussed in relation to the present findings.

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine.

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with "pA2" values in the rat isolated uterus assay of approximately 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA2 values of 6.7, 5.8, and less than 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and less than 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar1, Cha8]ANG II (7%), [Aib1, Cha8]ANG II (12%) and [Des1, Cha8]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.

Effect of constant light exposure on circulating gonadotrophin levels and hypothalamic gonadotrophin-releasing hormone (GnRH) content in the ovariectomized pony mare.

Melatonin is thought to play a role in relaying photic information to the central nervous system as part of the seasonal reproductive cycle of the mare. However, the mechanisms by which melatonin may act are unknown. Therefore, this study was designed to determine whether exposure to constant light would, by reducing circulating melatonin concentrations, have any effect on hypothalamic gonadotrophin-releasing hormone (GnRH) content and circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Blood samples were collected for 12 h at 15-min intervals from 8 ovariectomized (OVX) pony mares under ambient light conditions (12 h light, 12 h dark, October 6). Animals were then placed under representative ambient light (12 h light, 12 h dark; control, n = 4) or constant light (24 h light, 0 h dark; treatment, n = 4) in light-controlled rooms. Blood samples were collected daily and on experimental Days 14, 21 and 28 samples were collected at 15-min intervals for 12 h for analysis of circulating LH and melatonin (Bleed 2, 3 and 4, respectively). All animals were killed on Day 28 (following Bleed 4) and the hypothalami were collected. Mares exposed to constant light had significantly higher (P less than 0.05) LH concentrations in daily blood samples and showed significantly (P less than 0.05) higher LH concentrations during frequent sampling periods on Days 14 and 21 (Bleeds 2 and 3) compared with control mares. FSH did not differ significantly among groups in the daily samples. GnRH content was 1.5994 +/- 0.325 and 0.9457 +/- 0.193 pg/mg protein (treatment and control respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin 'antipeptides': (-)messenger RNA complementary to human angiotensin II (+)messenger RNA encodes an angiotensin receptor antagonist.

(-)mRNA complementary to human angiotensin II (+)mRNA encodes the 'antipeptide' Glu-Gly-Val-Tyr-Val-His-Pro-Val which is structurally related to angiotensin II. Angiotensin II 'antipeptide' (antiANG II) and the desglutamyl heptapeptide (antiANG III) are Type I antagonists which inhibit the contractile action of angiotensin at smooth muscle receptors by binding to a negative modulatory site on the angiotensin receptor which is distinct from the angiotensin binding site. These findings may illustrate that the inhibitory binding site on the angiotensin receptor exists to accomodate a naturally occurring inhibitor(s), which is encoded by the DNA strand complementary to that encoding angiotensin II.

Inhibition of oxytocin-induced but not angiotensin-induced rat uterine contractions following exposure to sodium sulfide.

Low concentrations (0.15-15 microM) of sodium sulfide reversibly attenuated the contractile response of the isolated rat uterus to oxytocin without affecting angiotensin II responsiveness. These findings suggest that functionally important disulfide bonds in the rat uterine oxytocin receptor, but not the angiotensin receptor, are sensitive to hydrosulfide ion. Reduction of oxytocin receptors by hydrosulfide ion may be a mechanism by which low levels of H2S delay parturition in rats.

Importance of the N-terminal domain of the type II angiotensin antagonist sarmesin for receptor blockade.

Analogues of the competitive angiotensin antagonist [Sar1,Tyr(ME)4]angiotensin II (sarmesin) with modifications at the N-terminus have been prepared by the solid-phase method and purified by reversed-phase HPLC. Substitution of the Sar1 residue of sarmesin with N,N-dimethyl-Gly, N-ethyl-Gly, aminoisobutyric, (methylamino)isobutyric, aminocaproic, and oxamic acids gave analogues that had the following respective antagonist activities (pA2) in the rat isolated uterus assay: less than 6, 6.9, 5.5, 6.0, less than 6, and 5.3. The additional substitution of Ile for Phe at the C-terminus of the latter four peptides gave pA2 values of 7.1, 5.1, less than 5, and 5. Substitution of the Arg2 residue of sarmesin with Nle or Sar abolished antagonist activity. These data emphasize the stringent and discriminating structural requirements in the N-terminal domain of sarmesin that endow this analogue with its antagonist properties and suggest the presence of defined steric constraints in this region of the molecule during receptor blockade.

Structure-activity relationships for the competitive angiotensin antagonist [sarcosine,O-methyltyrosine4]angiotensin II (sarmesin).

Analogues of the competitive angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (sarmesin) in which the sarcosine-1, O-methyltyrosine-4, and phenylalanine-8 residues were modified have been synthesized by the solid-phase method. The agonist and antagonist potencies of the 23 peptides synthesized were determined in the rat isolated uterus assay. At position 1, replacement of Sar with Asp, Ala, or Pro gave inactive analogues, and deletion of the N-terminal amino acid produced inactive heptapeptides for all analogues investigated. At position 4, substitution of Tyr with Tyr(Et), D-Tyr, D-Phe, Ile, Thr, or Hyp resulted in inactive analogues, whereas substitution of Phe gave a potent competitive antagonist (pA2 = 7.9), which retained significant agonist activity (22%). For position 8, [Sar1,Tyr(Me)4,Ile8]ANG II and [Sar1,Phe4,Ile8]ANG II were weaker antagonists (pA2 = 6.6 and 6.7, respectively) than [Sar1,Ile8]ANG II (pA2 apparent = 8.1) and, moreover, were reversible competitive antagonists. These findings demonstrate that the structural requirements for receptor blockade by sarmesin are remarkably stringent--modifications at positions 1, 4, and 8 markedly reduce the antagonist activity of this peptide.

Structure-desensitization relationships of angiotensin analogues in the rat isolated uterus.

The desensitizing potencies of angiotensin II (ANG II) analogues modified at positions 1, 2, 4, 7, and 8 have been examined in the rat isolated uterus assay by determining the time of recovery of the half-maximal concentration (EC50) response to angiotensin II after treatment of the tissues with a high dose (10(-5) M) of each analogue for 2 min. The magnitude of the desensitization effect was substituent dependent in the following manner: position 1, sarcosine (Sar) greater than Asp greater than des-Asp; position 2, Arg greater than Sar; position 4, Tyr greater than Tyr(Me) approximately Phe; position 7, 3,4-dehydroproline (Dpr) greater than Pro greater than thioproline (Tpr) greater than Sar; position 8, Ile greater than D-Trp greater than Ala greater than Phe. The "additivity" rule applied to these structure-desensitization relationships and the most potent desensitizer, requiring 3 h for reestablishment of the EC50 response, was [Sar1, Dpr7, Ile8]-ANG II. The desensitizing potencies of these analogues did not correlate with agonist or antagonist activities and demonstrated that the angiotensin-mediated tissue desensitization process has unique structural determinants. Methylation or elimination of the tyrosine hydroxyl group of strong desensitizers virtually eliminated the desensitization effect, implicating the phenoxyl moiety in the mechanism of desensitization. The initial phase of recovery of angiotensin responsiveness after desensitization by several analogues appeared to obey first-order kinetics. The results are discussed in the contexts of both one- and two-site receptor models.

Synthesis and biological activities of analogues of angiotensins II and III containing O-methyltyrosine and D-tryptophan.

Analogues of angiotensin II and III (ANG II and ANG III) in which the tyrosine and/or phenylalanine residues were substituted have been synthesized by the solid-phase method and purified by (carboxymethyl)cellulose chromatography and reversed-phase HPLC. The antagonist and agonist potencies of these peptides were determined in the rat isolated uterus assay. [Sar1,Tyr(Me)4]ANG II, [Tyr(Me)3]ANG III, [Sar1,D-Trp4]ANG II, [D-Trp3]ANG III, [Sar1,D-Trp8]ANG II, [D-Trp7]ANG III, [Sar1,Tyr(Me)4,Ile8]ANG II, [Tyr(Me)3,Ile7]ANG III, [Sar1,D-Trp4,Ile8]ANG II, [D-Trp3,Ile7]ANG III, [Sar1,Tyr(Me)4,D-Trp8]ANG II, and [Tyr(Me)3,D-Trp7]ANG III had antagonist activities (pA2) respectively of 8.1, less than 6, less than 6, less than 6, (7.7), (6.7), 7.2, less than 6, less than 6, less than 6, 7.1, and less than 6. The agonist activity of each peptide was less than 0.1% of that of ANG II. Analogues in which only the Phe residue was substituted were not readily reversible in the bioassay, whereas analogues in which only the Tyr residue or both the Tyr and Phe residues were substituted were reversible antagonists. Peptides that were twice substituted had lower antagonist activities than peptides having a single aromatic residue substitution. Substitution of the Tyr residue in ANG II, but not ANG III, provides a new route for the synthesis of potent and competitive angiotensin antagonists. Differences in the biological properties of ANG II and ANG III analogues substituted at the Tyr residue suggest different binding/conformation requirements for the two endogenous ligands at angiotensin receptors in smooth muscle.

A new approach to angiotensin antagonists: methylation of the tyrosine hydroxyl in angiotensin II.

[Sar1, Tyr(Me)4]angiotensin II, synthesized by the solid phase method and purified by ion-exchange chromatography and reversed-phase HPLC, was found to inhibit the contractile response to angiotensin II in the rat isolated uterus and inhibit the pressor response to angiotensin II in the vagotomized ganglion-blocked rat. In the rat isolated uterus Schild plots gave a pA2 of 8.1, and a slope of 0.9 indicative of competitive inhibition. In the rat pressor assay, infusion of the analogue at a rate of 500ng/kg/min caused a parallel displacement of the dose-response curve to ANG II to the right. In contrast, the classical angiotensin inhibitor [Sar1, Ile8] ANG II appeared to demonstrate non-competitive inhibition in both the rat isolated uterus and the pressor assays. The phenolic hydroxyl of phenoxide anion of Tyr4 in angiotensin II appears to be critical for the activation of angiotensin receptors in smooth muscle. Alkylation of the tyrosine residue in angiotensin analogues provides a new route for the synthesis of potent competitive antagonists of angiotensin.

Chemical and in vivo studies of the anion oxo[N,N'-ethylenebis(2-mercaptoacetimido)]Technetate(V).

Studies of the anionic coordination complex 99Tc-oxo[N,N'-ethylene-bis(2-mercaptoacetimido)]technetate(V) ([TcO(ema)]-) are described. Syntheses performed both at carrier levels (10(-5)M) and with no carrier added (less than 10(-8)M) indicate that the complex is formed virtually quantitatively from pertechnetate ion over this range. Tissue distributions in normal rats are similar at both concentrations up to one hour after administration. It has been shown--using a combination of high-pressure liquid chromatography and field-desorption mass spectrometry--that the anion is excreted unchanged into both urine and bile. The effectiveness of this N2S2 donor set in sequestering Tc-99m, and the in vivo stability of the resulting complex, suggest that modified chelates of this structural class could provide a series of useful diagnostic agents.