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Biomedical subjects

V J Hruby

Publications and source records attributed to V J Hruby.

At least 397 records · Page 22Linked to original sources
PubMed1984

Roles of mu, delta and kappa opioid receptors in spinal and supraspinal mediation of gastrointestinal transit effects and hot-plate analgesia in the mouse.

The opioid receptors involved in the mediation of thermal analgesia (55 degrees C hot-plate) and inhibition of gastrointestinal transit at the spinal and supraspinal levels were studied in unanesthetized mice. Five receptor-selective compounds were evaluated for effectiveness in eliciting analgesia and inhibiting transit after both i.c.v. and intrathecal administration; these included the proposed mu agonist, [D-Ala2, N-methyl-Phe4, Gly5-ol]enkephalin (DAGO), the proposed delta agonists, [D-Pen2, L-Pen5]enkephalin (DPLPE), [D-Pen2, D-Pen5]enkephalin (DPDPE) (conformationally constrained delta selective enkephalin analogs) and [D-Thr2, Thr6, Leu5]enkephalin (DTTLE), and the proposed kappa agonist, trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolidinyl)-cyclohexyl]- benzeneacetamide methanesulfonate (U-50,488H), as well as the nonselective mu-acting agonist, morphine. All compounds were found to produce analgesia after i.c.v. administration; the rank order of potency by the i.c.v. route was DAGO greater than DTTLE greater than morphine greater than DPLPE greater than DPDPE greater than U-50,488H. The analgesic effectiveness of most of these agonists given i.c.v. was evident for up to 40 min, with only DTTLE and U-50,488H having briefer time courses. Similarly, all the compounds produced analgesic responses after intrathecal administration, with the rank order of potency by this route being DTTLE greater than morphine greater than DAGO greater than DPLPE greater than DPDPE greater than U-50,488H, and all compounds (except U-50,488H) had durations of action of up to 20 to 40 min. These agonists also inhibited gastrointestinal transit after intrathecal administration, with a rank order of potency of DAGO greater than DTTLE greater than DPLPE greater than morphine greater than DPDPE greater than U-50,488H.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1984

Cerebral delta opioid receptors mediate analgesia but not the intestinal motility effects of intracerebroventricularly administered opioids.

Conformationally constrained cyclic enkephalin analogs which possess a high selectivity for the delta opioid receptor were used to determine the relative contribution of mu and delta receptors to brain-mediated changes in small intestinal propulsion and increases in hot-plate response time. Receptor preferences were determined by comparing the relative potencies of several opioid agonists in suppressing the electrically evoked contractions of the guinea-pig ileum and mouse vas deferens preparations. The ratio of IC50 values obtained in the guinea-pig ileum and the mouse vas deferens was used as an index of delta receptor selectivity. Effects on intestinal transit were determined in rats in which a silastic cannula had been implanted in the proximal duodenum and a polyethylene cannula in the right lateral cerebral ventricle (i.c.v.). Movement of a radioactive marker along the length of the small intestine after instillation into the duodenum was used to evaluate drug-induced changes in intestinal transit. The analgesic effects of i.c.v. administered opioids were determined in a second group of rats in which i.c.v. cannulas alone had been implanted. After i.c.v. administration of the agonist, the rats were placed on a 55 degrees C hot plate and the latency to rear paw-lick was timed. Compounds which showed a preference for the mu receptor [( D-Ala2, N-methyl-Phe4, Gly5 -ol]enkephalin and morphine/normorphine) were the most potent agonists at producing thermal analgesia and inhibition of small intestinal transit, whereas nonselective compounds (beta-endorphin and [D-Ala2, Met5]enkephalinamide) were slightly less potent in these assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesia↗
PubMed1984

Cyclic melanotropins. Part VII: Modified ring structures--synthesis and biological activity.

The highly potent cyclic analogue of alpha-MSH, Ac-[Cys4,Cys10]-alpha-MSH4-13-NH2, was structurally modified in position 4. Four analogues were prepared and their biological activities in the in vitro frog and lizard skin bioassays were determined. It was shown that removing the terminal acetylamino group to give [Mpa4,Cys10]-alpha-MSH4-13-NH2 resulted in little change in the biological activity, but a change in the stereochemistry of cysteine in position 4 to give Ac-[D-Cys4,Cys10[-alpha-MSH4-a3-NH2 led to a small decrease of activity in both bioassays. Decreasing the size of the intramolecular ring by removing one methylene group to give [Maa2,Cys10]-alpha-MSH4-13-NH2, resulted in an analogue with lower activities in both assays (about 3 times in the lizard and 500 times in the frog), and increasing the size of the righ by methylene group to give Ac-[Hcy4,Cys10]-alpha-MSH4-13-NH2 led to much lower activities in the lizard system and similar effects were seen upon decreasing the ring size in the frog skin assay.

Amino Acid Sequence↗
PubMed1983

Re-evaluation of glucagon1-6: the N-terminal hexapeptide of glucagon is not biologically active in the hepatic adenylate cyclase system.

The N-terminal hexapeptide of glucagon and the corresponding carboxamide analog, were prepared by solid-phase synthesis and tested for biological activity in the hepatic adenylate cyclase system. Both peptides were found to be inactive, even at concentrations of 10 mM. The differences observed in the activity of our compounds compared to previous reports, is ascribed to the presence of a contaminant found in earlier preparations which activates adenylate cyclase.

Adenylyl Cyclases↗
PubMed1983

Conformationally constrained cyclic enkephalin analogs with pronounced delta opioid receptor agonist selectivity.

The enkephalin analogs, [D-Pen2,L-Cys5]- and [D-Pen2,D-Cys5]-enkephalin are cyclic compounds, conformationally constrained by virtue of their 14-membered, disulfide containing rings and by the rigidizing effect of the beta, beta dimethyl substituents of the penicillamine side chain. The analogs exhibit profound delta receptor specificity as assessed by their relative potencies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, exhibiting, respectively, 666 and 215 times higher potency in the latter assay system. By contrast, the receptor selectivities measured in rat brain binding assays in the absence of sodium were much more modest, the cyclic analogs being, respectively, 15.2 and 6.0 times more effective at displacing [3H] [D-Ala2,D-Leu5]enkephalin than [3H]naloxone. However, for binding assays performed in the presence of a sodium concentration equivalent to that used in the GPI and MVD assays, these binding selectivities increased to 167 and 49, respectively.

Animals↗
PubMed1983

Cyclic penicillamine containing enkephalin analogs display profound delta receptor selectivities.

The cyclic, penicillamine(beta, beta dimethylcysteine)-containing enkephalin analogs, [D-Cys2, L-Pen5]-and [D-Cys2, D-Pen5]enkephalin and the corresponding bis-penicillamine analogs, [D-Pen2, L-Pen5]-and [D-Pen2, D-Pen5]enkephalin were synthesized and evaluated for opioid activity in the guinea pig ileum (GPI) and mouse vas deferens (MVD) bioassays and in rat brain and neuroblastoma-glioma cell membrane binding assays. These analogs all displayed delta receptor selectivity as assessed by IC50(GPI)/IC50(MVD) ratios and by their relative potencies for displacing [3H]naloxone (NAL) vs. [3H] [D-Ala2, D-Leu5]enkephalin (DADLE) from rat brain membrane preparations. For [D-Pen2, L-Pen5]- and [D-Pen2, D-Pen5]enkephalin the observed IC50(GPI)/IC50 (MVD) ratios (1088 and 3164) and IC50NAL/IC50DADLE ratios (371 and 175) represent a vast improvement over previously reported delta receptor selective ligands.

Animals↗
PubMed1983

A comparison of the analgesic and gastrointestinal transit effects of [D-Pen2, L-Cys5]enkephalin after intracerebroventricular and intrathecal administration to mice.

Intrathecal (i.t.) administration of the highly delta selective peptide, [D-Pen2, L-Cys5]enkephalin (DPLCE) (1-10 micrograms), effectively inhibited gastrointestinal transit of an orally-given radiolabelled marker in mice. By contrast, the same doses did not affect marker transit after intracerebroventricular (i.c.v.) administration. I.c.v. or i.t. administration of the peptide effectively increased the latency to hindpaw lick using the 55 degrees C hot-plate as the nociceptive stimulus. Maximum analgesic effects were seen with 0.3 micrograms given i.t. or 10 micrograms given i.c.v. Time-response studies showed activity for as long as 20 min after administration by either route. The differential gastrointestinal effects of DPLCE after i.c.v. and i.t. administration to mice suggest that delta receptors in the brain may mediate analgesic but not gut effects while spinal cord receptors may be less functionally selective.

Analgesia↗
PubMed1983

Bis-penicillamine enkephalins possess highly improved specificity toward delta opioid receptors.

The conformationally restricted, cyclic, disulfide-containing, enkephalin analogs [2-D-penicillamine, 5-L-penicillamine]enkephalin [(D-Pen2,L-Pen5]enkephalin) and [2-D-penicillamine, 5-D-penicillamine]enkephalin [(D-Pen2,D-Pen5]enkephalin) were synthesized by solid-phase methods. Selectivities of these analogs for a single class of opioid receptor were investigated by examining relative potencies in the mouse vas deferens assay, in which the functional receptor is the delta receptor, versus the guinea pig ileum assay, in which the mu receptor is the functional receptor, and by determining their relative abilities to displace the prototypical delta receptor ligand [D-Ala2, D-Leu5]enkephalin and the prototypical mu receptor ligand naloxone from rat brain membrane preparations. Based on these comparisons [D-Pen2,L-Pen5]- and [D-Pen2,D-Pen5]enkephalin exhibited delta receptor selectivities of 1,088 and 3,164, respectively, in the bioassays, and 371 and 175, respectively, in the binding assays. Compared with the previously reported delta receptor selective analogs, [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, and [D-Thr2,Leu5,Thr6]enkephalin, the bis-Pen-containing analogs provide an order of magnitude increase in delta receptor selectivity.

Animals↗
PubMed1983

Pharmacological, conformational and dynamic properties of cycloleucine-2 analogues of oxytocin and [1-penicillamine]oxytocin.

The solid phase syntheses of [2-cycloleucine] oxytocin and [1-penicillamine, 2-cycloleucine] oxytocin are reported. [1-Penicillamine, 2-cycloleucine] oxytocin is an oxytocin antagonist exhibiting no in vitro oxytocic activity. In the in vitro oxytocic assay, [1-penicillamine, 2-cycloleucine] oxytocin has a pA2 value of 6.70 +/- 0.08. [2-Cycloleucine]-oxytocin is a full oxytocin agonist exhibiting 4.9 +/- 0.5 U/mg of oxytocic activity. Neither compound possesses any measurable agonist or antagonist activity in the rat pressor assay. Carbon-13 nuclear magnetic resonance chemical shift parameters and spin-lattice relaxation times (T1) of the antagonist, [1-penicillamine, 2-cycloleucine] oxytocin, indicate that the antagonist exhibits similar conformational and dynamic properties as other oxytocin inhibitors previously studied. The carbon-13 nuclear magnetic resonance shift parameters and spin-lattice relaxation times (T1) of the oxytocin agonist, [2-cycloleucine] oxytocin, indicate that the agonist exhibits similar conformational and dynamic properties as oxytocin. These results are discussed in terms of the different receptor requirements for agonist and antagonist activities. It appears that there are different structural and conformational requirements at the 2-position for oxytocic agonist and antagonist activities.

Animals↗
PubMed1982

Conformational restrictions of biologically active peptides via amino acid side chain groups.

Determining the relationships between conformation and biological activity in peptide hormones and neurotransmitters is an important goal of contemporary biology. A major difficulty in these studies is the conformational flexibility of most peptides and the high dependence of the conformations on environment. The question arises whether conformations determined in solution are relevant to those important to the peptide at the membrane receptor(s). One recent approach to overcome these difficulties has been the use of conformational constraints by covalent bonding of side chain groups of residues in the peptide. In this manner linear peptides are rendered cyclic, and cyclic peptides are further conformationally constrained either by ring contractions or by other conformational constraints. Biologically active peptides specifically designed by this approach have been found to possess several useful properties including: 1) greater conformational integrity; 2) increased agonist or antagonist potency; 3) prolonged biological activity; 4) increased enzymatic stability; and 5) increased specificity for a particular receptor. Careful applications of this approach have provided important new designs features for peptide structure-function studies, and new insights into peptide conformation-activity relationships for oxytocin, somatostatin, enkephalin, bradykinin, vasopressin, and other biologically active peptides.

Hormones↗
PubMed1982

Conformational comparisons of oxytocin agonists, partial agonists, and antagonists using laser Raman and circular dichroism spectroscopy. Examination of 1-penicillamine and diastereoisomeric analogues.

The biological activity of peptide hormones and analogues depends on the structural and conformational properties of these compounds. A comparative study of the conformational properties of diastereoisomeric analogues of oxytocin with weak agonist activities (fully active but low potency), partial agonist activity (only able to partially induce biological response), and of conformationally restricted 1-penicillamine analogues with potent antagonist activity (no intrinsic activity, but can block the hormone's activity) was made using circular dichroism and laser Raman spectroscopies. Conformational information regarding the peptide amide, disulfide, and tyrosine chromophores was obtained, and indicates differences in the hormone agonists and antagonists. The diastereoisomeric oxytocin analogues [1-hemi-D-cystine]-, [2-D-tyrosine]-, and [5-D-asparagine]-oxytocin, have spectral features consistent with overall backbone conformations similar to oxytocin itself, but with differences in side chain moieties. This suggests that the substantial decrease in potency of the diastereoisomeric oxytocin analogues is due to changes in the relative orientations of the side chains. In contrast, the 1-penicillamine analogues of the present study, [1-penicillamine, 4-threonine]- and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin, like 1-penicillamine oxytocin analogues previously examined, have different backbone and disulfide conformations than oxytocin. All the 1-penicillamine oxytocin derivatives thus far examined appear, from laser Raman and CD data, to have similar topologies. However, those of the present study seem to have more rigid conformations as evidenced by very intense amide n-pi* and tyrosine pi-pi* CD transitions.

Circular Dichroism↗
PubMed1982

Structure-conformation-activity studies of glucagon and semi-synthetic glucagon analogs.

Examination of glucagon structure-activity relationships and their use for the development of glucagon antagonists (inhibitors) have been hampered until recently by the lack of high purity of semisynthetic glucagon analogs and inadequate study of full dose-response curves for these analogs in sensitive bioassay systems. Recently a number of highly purified glucagon fragments and semi-synthetic analogs have been prepared and their full dose-response activities examined over a wide concentration range using the hepatic membrane adenylate cyclase assay, the hepatic membrane receptor binding assay, and glycogenolytic activity in isolated rat hepatocytes. The results of these studies have enabled us to identify and dissociate the structural (and in some cases conformational) features of glucagon important for binding from those most responsible for biological activity (transduction). Key findings in these studies were the observation that: (1) the C-terminal region of glucagon is primarily of importance for hormone binding to receptors; (2) glucagon 1-21 and glucagon 1-6 have low potency, but are essentially fully active glucagon derivatives; and (3) highly purified glucagon 2-29 ([1-des-histidine]-glucagon), [1-N alpha-carbamoylhistidine]-glucagon and [1-N alpha-carbamoylhistidine, 12-N alpha-carbamoyllysine]-glucagon are all partial agonists. These and other findings led us to synthesize several semisynthetic analogs of glucagon which were found to possess no intrinsic biological activity in the hepatic adenylate cyclase assay system, but which could block the effect of glucagon (competitive inhibitors) in activating adenylate cyclase in this system. Two of these highly purified analogs [1-des-histidine][2-N alpha-trinitrophenylserine, 12-homoarginine]-glucagon and [1-N alpha-trinitrophenylhistidine, 12-homoarginine]-glucagon were quite potent glucagon antagonists (inhibitors) with pA2 values of 7.41 and 8.16 respectively. The latter compound has also been demonstrated to decrease dramatically blood glucose levels of diabetic animals in vivo. These results demonstrate that glucagon is a major contributor to the hyperglycemia of diabetic animals. Examination of the known and calculated conformational properties of glucagon provide insight into the structural and conformational properties of glucagon and its analogs most responsible for its biological activity. Consideration of these features and the mechanism of glucagon action at the membrane receptor level provide a framework for further developing glucagon analogs for theoretical and therapeutic applications.

Adenylyl Cyclases↗
PubMed1982

Hyperglycemia of diabetic rats decreased by a glucagon receptor antagonist.

The glucagon analog [l-N alpha-trinitrophenylhistidine, 12-homoarginine]-glucagon (THG) was examined for its ability to lower blood glucose concentrations in rats made diabetic with streptozotocin. In vitro, THG is a potent antagonist of glucagon activation of the hepatic adenylate cyclase assay system. Intravenous bolus injections of THG caused rapid decreases (20 to 35 percent) of short duration in blood glucose. Continuous infusion of low concentrations of the inhibitor led to larger sustained decreases in blood glucose (30 to 65 percent). These studies demonstrate that a glucagon receptor antagonist can substantially reduce blood glucose levels in diabetic animals without addition of exogenous insulin.

Animals↗