Search PubMed⌕ Search

Biomedical subjects

V J Hruby

Publications and source records attributed to V J Hruby.

At least 217 records · Page 12Linked to original sources
PubMed1992

Synthesis and binding characteristics of the highly delta-specific new tritiated opioid peptide, [3H]deltorphin II.

A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a Kd value of 1.9 nM, and the Bmax was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10,000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species.

Analgesics↗
PubMed1992

Antinociceptive interactions of opioid delta receptor agonists with morphine in mice: supra- and sub-additivity.

In this study, the antinociceptive interactions of fixed ratio combinations of intracerebroventricularly (i.c.v.) given morphine and subantinociceptive doses of the delta agonists, [D-Pen2, D-Pen5]enkephalin (DPDPE), [D-Ala2, Glu4]deltorphin (DELT) or [Met5]enkephalin (MET) were examined using the mouse warm water tail flick test. When morphine was coadministered with DPDPE or DELT in a 4:1 and 9:1 mixture, respectively, a synergistic antinociceptive effect was observed. In contrast, when morphine was coadministered with MET in a 1:2 fixed ratio mixture, a subadditive interaction occurred. These results demonstrate both positive and negative modulatory interactions of delta agonists with morphine in an antinociceptive endpoint and that these interactions can be either supra- or subadditive. The data support the concept of a functional interaction between opioid mu and delta receptors and a potential regulatory role for the endogenous ligands of the opioid delta receptor.

Analgesics↗
PubMed1992

Synthesis and bioactivity studies of two isosteric acyclic analogues of melanin concentrating hormone.

Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide. MCH stimulates perinuclear aggregation of melanosomes within integumental melanocytes of teleost fishes resulting in skin blanching. MCH contains a disulfide bridge forming a 10-residue ring [sequence: see text]. It has been proposed that the ring is necessary for maintenance of potency. In order to test this proposal, we have synthesized two pseudo-isosteric analogues of MCH that cannot cyclize. They differed only in the polarity of the side chain group of positions 5 and 14. Serine was substituted for Cys5 and Cys14 in one peptide and L alpha-aminobutyrate (Abu) was the substitution at the two positions in the other peptide. Using a fish skin bioassay we determined that these analogues exhibit less than 1/10,000th the potency of the native hormone. These results suggest that the disulfide bridge is necessary to maintain the correct conformational and topographical features of the hormone for receptor binding and transmembrane signal transduction.

Amino Acid Sequence↗
PubMed1992

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.

Animals↗
PubMed1992

The metabolic effects of oxytocin are mediated by a uterine type of receptor and are inhibited by oxytocin antagonist and by arginine vasopressin in the dog.

Infusion of oxytocin (OT) into normal dogs, in doses which produced plasma levels of OT in the physiological range, has been shown to increase plasma levels of glucose, insulin and glucagon and increase rates of glucose production and uptake. This study sought to determine whether there was a correlation between these metabolic effects and the oxytocic potency of four less potent oxytocic analogues when infused into normal dogs. The rank order of oxytocic potency of all 4 correlated well with the rise in plasma glucose levels, and in 3 of the 4 with the rise in plasma insulin levels. An antagonist of the oxytocic effect of OT suppressed the usual OT-induced rise in plasma glucose, insulin and glucagon as well as the increased glucose production and uptake. Arginine vasopressin (AVP) infusion, which by itself did not produce any metabolic effects, blocked completely the effects of OT infusion to raise plasma glucose and insulin levels and increase glucose production and uptake. The data suggest that the metabolic effects of OT in the dog are mediated by OT receptors that are similar to those producing the oxytocic effects. Whether the inhibition by AVP of the metabolic and hormonal effects of OT occurs at the receptor or post receptor level or via other mechanisms remains to be determined.

Animals↗
PubMed1992

Topographical requirements for delta opioid ligands: common structural features of dermenkephalin and deltorphin.

We propose a common topographical model for the bioactive conformation of deltorphin and dermenkephalin at the delta opioid receptor. In this model a hydrophilic surface from the N- to C-termini is surrounded by lipophilic residues ("hot dog" structure). The important element that orients the N-terminal tyramine is the interaction of the N-terminal amino group, with the carboxyl group of Asp4 in deltorphin I and with Asp7 through His4 (as a triad) in dermenkephalin. The biological properties of synthetic analogues designed to test this model demonstrate that the hydrophilic amino acid residues of these peptides are interchangeable. In addition, incorporation of Aib residues that change the lipophilic topography of these molecule, strongly reduces affinity for the delta opioid receptor.

Amino Acid Sequence↗
PubMed1992

Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.

Animals↗
PubMed1992

Enzymological studies of melanin concentrating hormone (MCH) and related analogues.

1. Salmon melanin concentrating hormone (MCH) is a cyclic heptadecapeptide possessing the following primary structure: Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val. 2. In the fish, Synbranchus marmoratus, skin bioassay MCH5-15 is equipotent to MCH whereas MCH5-14, which comprises only the ring structure, is about 100-fold less active. 3. MCH and two fragment analogues, MCH5-15 and MCH5-14, were studied to determine their relative stability in the presence of fish serum and purified proteolytic enzymes, trypsin and alpha-chymotrypsin. 4. After 4 hr incubation in fish serum, MCH5-15 retained 1/100, MCH5-14 1/1000 and MCH only 6/1000 of the potency of the native hormone. 5. The three peptides were also very resistant to degradation by purified proteolytic enzymes involving the following relative order of resistance: MCH5-14 > MCH5-15 > MCH. MCH5-14 potency was not altered after a 1 hr incubation in either enzyme whereas MCH retained 1/10 and 4/100 of its original potency, and MCH5-15 retained 1/10 and 8/10 of its original potency, after 1 hr in trypsin and alpha-chymotrypsin, respectively.

Amino Acid Sequence↗
PubMed1992

Synthesis and biological activities of fatty acid conjugates of a cyclic lactam alpha-melanotropin.

Four fatty acid conjugates of a cyclic lactam-bridged alpha-MSH fragment analogue were synthesized and their potencies and biological activities compared in several melanotropin bioassays. Palmitoyl, myristoyl, decanoyl, and hexanoyl conjugates of H-Asp-His-D-Phe-Arg-Trp-Lys-NH2 were prepared. In the in vitro mouse melanoma cell assay, each of the conjugates was 10-100 times more potent than alpha-MSH or the substrate peptide in elevating tyrosinase activity. The shorter conjugates of hexanoic and decanoic acid were as potent as alpha-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogues were about 100 times less potent. The potency of the myristoyl and palmitoyl conjugates increased with time in contact with the skins. These observations may be related to the more lipid-like nature of these peptide-fatty acid conjugates. Each of the conjugates exhibited prolonged melanotropic activity in the lizard skin bioassays and in the mouse S91 melanoma tyrosinase bioassay, since the biological response continued following removal of the conjugates from the incubation media. The prolonged residual melanotropic activity resulted from conjugation of the fatty acids to the MSH fragment analogue since the analogue itself did not exhibit prolonged activity.

Amino Acid Sequence↗
PubMed1992

Reduced peptide bond cyclic somatostatin based opioid octapeptides. Synthesis, conformational properties and pharmacological characterization.

The conformational and pharmacological properties that result from peptide bond reduction as well as the use of secondary amino acids in a series of cyclic peptides related to the mu opioid receptor selective antagonist D-Phe1-Cys2-Tyr3-D-Trp4-Orn5-Thr6-Pen7+ ++-Thr8-NH2 (IV), have been investigated. Peptide analogues that contain [CH2NH] and [CH2N] pseudo-peptide bonds (in primary and secondary amino acids, respectively) were synthesized on a solid support. Substitution of Tyr3 in IV by the cyclic, secondary amino acid 1,2,3,4-tetrahydroisoquinoline carboxylate (Tic) and of D-Trp4 with D-1,2,3,4-tetrahydro-beta-carboline(D-Tca4), gave peptides 4 and 1, respectively. Both analogues displayed reduced affinities for mu opioid receptors. Conformational analysis based on extensive NMR investigations demonstrated that the backbone conformations of 1 and 4 are similar to those of the potent and selective analogue D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (I), while the conformational properties of the side chains of Tic3 (4) and D-Tca4 (1) resulted in topographical properties that were not well recognized by the mu opioid receptor. Peptide bond modifications were made including (Tyr3-psi[CH2NH]-D-Trp4), 3; (Tyr3-psi[CH2N]-D-Tca4), 2; and (Cys2-psi[CH2N]-Tic3), 6. These analogues showed decreases in their mu opioid receptor affinities relative to the parent compounds IV, 1, and 4, respectively. 1H NMR based conformational analysis in conjunction with receptor binding data led to the conclusion that the reduced peptide bonds in 2, 3, 5, and 6 do not contribute to the process of discrimination between mu and delta opioid receptors, and in spite of their different dynamic behaviors (relative to 1 and 4), they are still capable of attaining similar receptor bound conformations, possibly due to their increased flexibility.

Amino Acid Sequence↗
PubMed1992

Conformationally biased analogs of oxytocin.

Four diastereomeric analogs of oxytocin containing substituted phenylalanine in position 2 were synthesized. This modified phenylalanine side chain contained one methyl group attached to the beta-carbon and the second one at the 2' position of the aromatic ring. All analogs were found to be inhibitors of uterotonic activity of oxytocin with pA2 values ranging from 6.0 to 8.3; the most potent one (pA2 = 8.3) contained dimethylphenylalanine of the D-erythro configuration.

Amino Acid Sequence↗
PubMed1992

Opioid receptor antagonists discriminate between presynaptic mu and delta receptors and the adenylate cyclase-coupled opioid receptor complex in the brain.

The present study addressed the question as to whether or not' interacting mu and delta opioid receptors, which may constitute an opioid receptor complex-inhibitory coupled to adenylate cyclase in rat neostriatum, display different antagonistic properties than the classical (noncomplexed) mu and delta receptors. In concentrations that antagonized the presynaptic inhibitory effect of [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO) on [3H]norepinephrine release from rat neocortical slices, the cyclic somatostatin-related mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 did not affect the inhibition of dopamine-sensitive adenylate cyclase caused by DAMGO in neostriatal slices. The delta opioid receptor antagonist naltrindole appeared to be about 200-fold more effective as an antagonist against inhibitory effect of [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 on [14C]acetylcholine release from neostriatal slices than against the inhibitory effect of DAMGO on [3H]norepinephrine release from neocortical slices, in agreement with the involvement of presynaptic delta and mu receptors, respectively. However, regarding the inhibitory effect of DAMGO and [D-Ser2(O-tert-butyl),Leu5] enkephalyl-Thr6 on adenylate cyclase activity in neostriatal slices, naltrindole not only displayed a very low affinity but also only 10-fold delta-selectivity. In striking contrast to D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 and naltrindole, naloxone did not discriminate between the neurotransmitter release-and adenylate cyclase-inhibitory effects of DAMGO and [D-Ser2(O-tert-butyl), Leu5]enkephalyl-Thr6.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1992

Whole body and brain distribution of [3H]cyclic [D-Pen2,D-Pen5] enkephalin after intraperitoneal, intravenous, oral and subcutaneous administration.

The route of administration of a given drug can have a significant influence upon whole body distribution. The present study examined whole body distribution of the delta opioid receptor-selective peptide [3H]DPDPE in male CD1 mice after administration by several routes. Additionally, we describe regional brain distribution of [3H]DPDPE after i.v. administration with and without pretreatment with naloxone or the selective delta receptor antagonist naltrindole. Finally, characterization of the inherent enzymatic stability of DPDPE was also examined. Intravenous administration results in a significantly large amount of [3H]DPDPE in the small intestine and flush at 15 and 30 min postadministration, suggesting rapid biliary excretion. The highest level in the brain after i.v. administration occurred at 60 min (0.08%). After i.p. and s.c. administration, large amounts of [3H]DPDPE were found in the small intestine and flush, but not until 60 min postadministration, suggesting a slower rate of absorption from the site of administration. The i.p. and s.c. groups' brain levels peaked at 120 min (0.07 and 0.09%, respectively). The highest levels in the brain after p.o. administration were seen at 240 min (0.03%). Examination of regional brain distribution data showed no significant difference in the levels of [3H]DPDPE between brain regions at any time point studied. However, naloxone pretreatment resulted in significant reductions of [3H]DPDPE in all brain regions at 5 and 10 min. Naltrindole pretreatment resulted in significant reductions in the frontal cortex and striatum at 5 and/or 10 min postadministration, but had no effect on [3H]DPDPE levels in cerebellum, hippocampus or brain stem.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1991

Induction of skin tanning by subcutaneous administration of a potent synthetic melanotropin.

UNLABELLED: OBJECTIVE--To determine the efficacy of short-term administration of a synthetic analogue of alpha-melanotropin, [Nle4D-Phe7] (NDP)-alpha-melanocyte-stimulating hormone (MSH), in darkening (tanning) human skin. DESIGN--Randomized, placebo-controlled, double-blind clinical trial. SETTING--Clinical research unit of a university medical center. SUBJECTS--Twenty-eight healthy white men with a history of either poor tanning (skin type I or II) or good tanning (skin type III or IV) recruited from advertisements and paid to participate in the study. METHODS--Each subject received 10 subcutaneous injections of either a purified NDP preparation or saline over 12 days. They were followed up for 7 weeks after therapy was completed. All subjects used a high-potency sunscreen during the trial. MAIN OUTCOME MEASURE--Skin darkening was quantified by serial chromaticity measurements prior to, during, and after therapy. RESULTS: -A significant parabolic curve of skin darkening activity was noted in subjects with skin type I or II (P less than .001) and with skin type III or IV (P much less than .001) who were given NDP. No darkening occurred in the subjects who were given a placebo. Peak changes were seen 1 to 3 weeks after therapy was completed. CONCLUSION--Human skin darkens as a response to a synthetic melanotropin given by subcutaneous injection. Skin tanning appears possible without potentially harmful exposure to ultraviolet radiation.

Adult↗
PubMed1991

Cyclic somatostatin analogues as potent antagonists at mu-, but not delta- and kappa-opioid receptors mediating presynaptic inhibition of neurotransmitter release in the brain.

The opioid receptor antagonist properties of four conformationally constrained cyclic octapeptide analogues of somatostatin were investigated using in vitro functional paradigms of mu-, delta- and kappa-opioid receptors in the rat brain. The analogues examined were D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), D-Tic-CTOP (TCTOP) and D-Tic-CTAP (TCTAP). Activation of mu-receptors by the enkephalin analogue Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAGO) inhibited the (electrically evoked) release of [3H]noradrenaline (NA) from superfused cortical slices and this inhibitory effect was antagonized in a competitive fashion by all of the octapeptides tested (pA2 values: CTOP and CTAP 7.9-8.0, TCTOP and TCTAP 8.7-8.8). Selective activation of kappa-opioid receptors by the cyclohexylbenzeneaceamide U69593 (0.02 microM) inhibited (by 40-45%) the release of [3H]dopamine (DA) from striatal slices, whereas selective activation of delta-opioid receptors by [D-Ser2(O-t-butyl),Leu5]enkephalyl-Thr6 (DSTBULET; 0.1 microM) caused an inhibition (by 38-46%) of striatal [14C]acetylcholine (ACh) release. However, these inhibitory effects were not affected by any of the octapeptides in concentrations that caused full antagonism of the inhibitory effect (55-65%) of 0.1 microM DAGO on cortical [3H]NA release. Thus, the cyclic octapeptide somatostatin analogues CTOP, CTAP, TCTOP and TCTAP are potent and highly selective antagonists at the mu-opioid receptors mediating presynaptic inhibition of NA release in the brain. The mu-receptor affinity of the most potent of these antagonists, TCTOP and TCTAP, appears to be similar to that of naloxone but these antagonists have a much greater selectivity than the latter.

Amino Acid Sequence↗
PubMed1991

Topographical requirements for delta opioid ligands: presence of a carboxyl group in position 4 is not critical for deltorphin high delta receptor affinity and analgesic activity.

To investigate the role of the carboxyl group in deltorphin molecules, we have synthesized three new analogues in which the acidic amino acid residues in position 4 of the deltorphins were replaced by non-acidic but hydrophilic amino acids residues. The three analogues, [Ser4]-, [Gln4]-, and [Cys4]-deltorphin, all are as potent or more potent than either deltorphin I or II at delta opioid receptors and possess good delta selectivities. The excellent correlation between their in vitro delta receptor potencies and their intrathecal antinociception activity forms a strong argument for involvement of those receptors in spinal nociceptive modulation in the rats.

Amino Acid Sequence↗
PubMed1991

High-performance liquid chromatography of peptides at reduced temperatures: separation of isomers.

Cholecystokinin analogues containing N-methyl amino acids were studied by reversed-phase high-performance liquid chromatography at reduced temperatures. A reduction in temperature to -17 degrees C led to lower efficiency, but at the same time separations of cis and trans isomers (and some impurities) were achieved. The velocity constants for cis-trans equilibria were calculated.

Amino Acid Sequence↗