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V J Hruby

Publications and source records attributed to V J Hruby.

At least 253 records · Page 14Linked to original sources
PubMed1988

Conformation of D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP-NH2), a highly selective mu-opioid antagonist peptide, by 1H and 13C n.m.r.

The 1H and 13C n.m.r. spectral parameters of CTP-NH2 [D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2], a potent, highly selective mu-opiate antagonist, were measured in aqueous solution and a possible conformation has been deduced from the spectral data. The data are consistent with a type II' beta-turn for the tetrapeptide sequence -Tyr3-D-Trp4-Lys5-Thr6-. Solvent shielding of the Cys2 amide proton, observed in variable temperature experiments, suggests an orientation of this amide proton toward the gem dimethyls of Pen7 with possible hydrogen bonding to the Thr6 carbonyl oxygen, and a dihedral angle of -110 degrees for the disulfide bond. Partially relaxed Fourier transform 13C relaxation studies confirm a constrained cyclic system, with the C alpha carbons in the "hinge" of the beta-turn having the shortest t1 times. Segmental motion was observed for the side chain of Lys5.

Carbon Isotopes↗
PubMed1988

Proton n.m.r. investigation of conformational influence of penicillamine residues on the disulfide ring system of opioid receptor selective somatostatin derivatives.

Three cyclic disulfide analogs related to somatostatin, D-Phe(1)-cyclo(Cys(2)-Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-Xxx(7))-Thr(8)- NH2 (where Xxx = L-Pen 1; L-Cys 3; or D-Pen 4) were examined in DMSO-d6 by one- and two-dimensional proton n.m.r. spectroscopy in order to analyze the conformational influence of the position-7 residue on the 20-membered disulfide ring. From these studies it was concluded that all three analogs maintain a beta II' turn solution conformation for the core tetrapeptide -Tyr(3)-D-Trp(4)-Lys(5)-Thr(6)-. However, the disulfide conformation differs in the analogs, with 1 and 3 having a left-handed and 4 a right-handed disulfide chirality.

Amino Acid Sequence↗
PubMed1988

Cholecystokinic activity of N alpha-hydroxysulfonyl-[Nle28,31]CCK26-33 analogues modified at the C-terminal residue.

Three new analogues of N alpha-hydroxysulfonyl-[Nle28,31]CCK26-33 are reported in which the C-terminal L-Phe33 residue has been replaced by L-Leu, D-Phe or N-methyl-L-Phe. Biological evaluation in a series of binding and bioassays demonstrates that both L-stereochemistry and an aromatic side chain at position-33 are essential for full agonist activity. While the L-Leu33 and D-Phe33 analogues had reduced potencies in stimulating contraction of the guinea pig ileum or gall bladder, the D-Phe33 analogue was fourfold selective for the ileum. This latter analogue also exhibited apparent partial agonism in the rat pancreatic amylase release assay. The N-methyl-L-Phe33 analogue was almost equipotent to the parent analogue in all bioassays, suggesting that this modification might be useful for introducing enzymatic stability in CCK analogues.

Acetylcholine↗
PubMed1988

Importance of the C-terminal alpha-helical structure for glucagon's biological activity.

The synthetic glucagon analogues [Glu21]glucagon, 2, and [Lys17,18,Glu21]glucagon, 3, were designed using Chou-Fasman calculations for the purpose of enhancing the probability for the formation of a C-terminal amphipathic alpha-helical conformation. Circular dichroism indicates increased alpha-helical content for these analogues in solution relative to glucagon. Analogues 2 and 3 also exhibit a 3-fold and 5-fold increase in receptor binding potency, respectively. The adenylate cyclase stimulating potencies of 2 and 3 relative to glucagon are 2.1 and 7 times greater, respectively. Attempts were made at further alpha-helical enhancement by further substitutions in the 10-13 region of glucagon, as represented by the glucagon analogues [Phe13,Lys17,18 Glu21]glucagon, 4, and [Phe10,13,Lys17,18,Glu21]glucagon, 5. These latter substitutions resulted in lowered receptor binding and adenylate cyclase potencies for 4 and 5 relative to 3 despite increased alpha-helical content in solution as observed by circular dichroism spectroscopy.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Body temperature response profiles for selective mu, delta and kappa opioid agonists in restrained and unrestrained rats.

In many cases, body temperature is altered in response to opioid agonists, but the direction, magnitude and time course of alteration vary with a number of factors. Body temperature may be subject to differential modification by different opioid receptor types. The authors examined the effect (i.c.v.) of the selective mu, delta and kappa opioid agonists, [D-Ala2, MePhe4, Gly5-ol] enkephalin (DAGO), [D-Pen2, D-Pen5] enkephalin and U50488H, respectively, on the body temperature of restrained and unrestrained rats. Each of the three opioid agonists produced a differentiable profile of body temperature changes. DAGO caused a primary decrease in body temperature of restrained rats and an increase in body temperature of unrestrained rats. The pretreatment dose of naloxone necessary to attenuate the hyperthermic response to DAGO of unrestrained rats was 10 times higher than that required to block the hypothermic response to DAGO in restrained rats. Low doses of both [D-Pen2, D-Pen5]enkephalin and U50488H caused a decrease in body temperature of both restrained and unrestrained rats. Hypothermic responses to U50488H were not blocked by naloxone, whereas hypothermic responses to [D-Pen2, D-Pen5]enkephalin in unrestrained rats were potentiated by naloxone. The results indicate that the three compounds modified body temperature by different means, suggesting activation of different opioid, and perhaps nonopioid, receptors. This may reflect a differential modulation of body temperature by endogenous opioids depending on the specific peptide released and the receptor type activated. Besides the physiologic implications, body temperature responses provided a sensitive pharmacologic measure for distinguishing the in vivo activity of different selective opioid agonists.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1988

Opioid receptor selectivity of beta-endorphin in vitro and in vivo: mu, delta and epsilon receptors.

The relative contributions of mu and delta opioid receptors in the response to Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe- Lys-Asn - Ala-Ileu-Ileu-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu (B-endorphin) were assessed as reductions in B-endorphin potency in the presence of mu and delta receptor selective antagonists in the guinea pig ileum, mouse vas deferens, rat vas deferens and in analgesic and gastrointestinal transit time tests in mice. We used the nonselective antagonist naloxone, the mu antagonist D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP) and the delta antagonist N,N,diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI 174,864) in each test system at concentrations that effectively antagonized the respective mu and delta agonists, Tyr-Pro-N-MePhe-D-Pro-NH2 and Tyr-D-Pen-Gly-Phe-D-Pen. In the guinea pig ileum, the inhibitory effects of 1 microM B-endorphin were blocked by 1 microM CTP and 1 microM naloxone, but not by 1 microM ICI 174,864. In the mouse vas deferens, B-endorphin (0.2 microM) was antagonized by 1 microM CTP, 1 microM ICI 174,864 and by 1 microM naloxone. In contrast, in the rat vas deferens, B-endorphin (0.01-1 microM) produced potent inhibitory actions that were blocked by 1 microM naloxone, but not by 1 microM-CTP or by 1 microM ICI 174,864. The mu agonist, Tyr-Pro-N-MePhe-D-Pro-NH2 (0.1-10 microM), like B-endorphin, also had inhibitory actions in the rat vas deferens, but its effects were blocked by 1 microM CTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Analgesia↗
PubMed1988

Natriuretic action of neurohypophysial peptides: effects of agonists and antagonists and implication of natriuretic receptor.

Neurohypophysial peptides possess natriuretic activity. Although it has been shown that the natriuretic action of these peptides can be dissociated from their antidiuretic activity (a V2-receptor mediated response), it is not known whether the V1-receptor or yet a third receptor type mediates the natriuretic response. Also, it has not been studied what effects V1- and V2-antagonists may have on urinary sodium excretion. To define this, we have studied the effects of four oxytocin (OT) agonists: arginine-vasopressin, OT, [Leu4]OT and [cyclo-Leu8]OT; two V1-receptor antagonists: [penicillamine1,Phe(Methyl)2,Thr4,Orn8]OT and [penicillamine1,D-Phe(Ethyl)2,Thr4,Orn8]OT and one V2-receptor antagonist: d-(CH2)5[D-Ile2,alpha-aminobutyric acid4]arginine-vasopressin on renal excretion of water and electrolytes in anesthetized rats under water diuresis. We also studied the effects of the antagonists on the OT-induced antidiuretic and natriuretic responses. Only the agonists, but not the antagonists, were found to have natriuretic activity. The natriuretic potency was not related to the peptide's antidiuretic activity, but was in the same rank order as their oxytocic activity (a V1-agonist effect). The effects of the antagonists on the OT-induced renal responses were studied at two dose levels, representing a strong and near maximal of their respective V1 and V2 inhibitory doses. The V1-antagonist had no effect on the antidiuretic response to OT but inhibited the natriuretic response in a dose-dependent manner. The antinatriuretic effect was also long-lasting as its antioxytocic activity. The V2-antagonist inhibited the antidiuretic response to OT in a dose-dependent manner but only the high dose inhibited the natriuretic response. These results indicate that the natriuretic action of OT was not mediated by V2-receptors and antinatriuresis was not specific for V1-antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Interactions of glucagon and glucagon analogs with isolated canine hepatocytes.

We have used glucagon and nine glucagon analogs to investigate the interactions of these ligands with glucagon-binding sites present on isolated canine hepatocytes. Curves reflecting the inhibition of 125I-labeled glucagon or 125I-labeled analog binding to cells by the 10 peptides spanned, overall, a 10(6)-fold range of hormone concentration, were consistent with hormone binding to two classes of binding sites in each case, and fell into two groups, one of which contained curves that were considerably more shallow than the other. Only conditions that emphasized prior binding to low affinity sites resulted in the rapid and extensive dissociation of receptor-bound ligand from isolated cells. Finally, all 10 peptides exhibited a concentration-dependent inhibition of the incorporation of [14C]fructose into hepatocyte glycogen that correlated best with dissociation constants for high affinity rather than for low affinity binding. We conclude that (a) the association of ligand with the high and low affinity glucagon-binding sites of isolated canine hepatocytes is a characteristic of analogs modified at diverse sites throughout the peptide hormone, (b) the different rates of dissociation of ligand from the two populations of binding sites most probably account for the biphasic dissociation of ligand from isolated cells and for the different affinities of the two receptor populations for ligand, and (c) the activity of glucagon and glucagon analogs to inhibit the incorporation of fructose into hepatocyte glycogen arises from the association of ligand with high affinity binding sites.

Animals↗
PubMed1987

Stimulation and inhibition of cAMP accumulation by glucagon in canine hepatocytes.

We have examined, by use of isolated canine hepatocytes and selected hormone analogs, the mechanisms by which glucagon modifies the accumulation of cellular cAMP. Low concentrations of glucagon (less than or equal to 3 nM) enhanced the accumulation of hepatocyte cAMP, whereas higher concentrations of the hormone diminished the effectiveness of lower ones. This biphasic concentration dependence was observed as well for some glucagon analogs, but not for others, and was apparent for cells incubated in the presence or absence of theophylline. Glucagon at high concentrations (greater than or equal to 10 nM) also inhibited the accumulation of cAMP induced by isoproterenol. The inhibitory effect of glucagon in both of these systems was reversed or attenuated by cell incubations involving the use of pertussis toxin (islet-activating protein) or a peptide antagonist of the glucagon-adenylyl cyclase system. We conclude that (a) glucagon, through its interaction with high and low affinity binding sites, can either stimulate or inhibit the production of hepatocyte cAMP; (b) the inhibitory action of the hormone appears to arise from interactions of ligand with a subset of these binding sites and to require structural characteristics in addition to those that determine receptor binding affinity per se; and (c) the glucagon and adrenergic systems involved in stimulating cAMP accumulation are linked, at least with regard to the negative effect induced by high concentrations of glucagon.

Adenylyl Cyclases↗
PubMed1987

Effects of nonapeptide antagonists on oxytocin- and arginine-vasopressin-induced analgesia in mice.

Several peptides, including arginine-vasopressin (AVP), neurotensin, and substance P, produce analgesia that is not mediated by opiate systems. Using the hot plate test, we studied the analgesic effects of intracisternal (i.c.) administration of various doses of the nonapeptide oxytocin (OXY) in Swiss-Webster mice. We found that OXY (1-4 micrograms) significantly increased the latency of animals to jump or lick their paws after placement on a hot plate. This effect was not blocked by naloxone pretreatment, which suggests that it is not opiate dependent. Using the hot plate test, we confirmed that AVP (1 and 4 micrograms) also produces analgesia. We then studied the analgesia produced by OXY and by AVP using 3 nonapeptide analogues with antagonist properties: [Pen1, LpMePhe2, Thr4, Orn8]OXY (PLMPTO-OXY) that has anti-oxytocic properties in the uterine contraction assay, d(CH2)5Tyr(Me)AVP(dTM-AVP) which antagonizes the antidiuretic properties of AVP and d(CH2)5D-Ile2,Abu4-AVP (dIA-AVP) which antagonizes the vasopressor effects of AVP. Simultaneous administration of PLMPTO-OXY completely blocked the analgesia produced by OXY whereas the antidiuretic antagonist dIA-AVP partially blocked OXY-induced analgesia and dTM-AVP had no effect. None of the antagonists used blocked AVP-induced analgesia. We concluded that the neural systems mediating the analgesic effects of i.c. OXY differ from those for AVP.

Analgesia↗
PubMed1987

Melanin concentrating hormone exhibits both MSH and MCH activities on individual melanophores.

Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val (melanin concentrating hormone, MCH) and several fragment analogs (MCH1-14, MCH5-17, MCH5-14) were synthesized and their biological activities determined in a very sensitive fish skin bioassay. The potency ranking and minimum effective doses of the peptides were determined to be: MCH1-17 (10(-12)M) greater than less than MCH5-17 (10(-12)M) greater than MCH1-14 (10(-11)M) greater than MCH5-14 (2 X 10(-10)M). The melanosome aggregating activity of MCH could be completely reversed by a 100-fold higher concentration of pounds-MSH. MCH was self-antagonized in a dose-related manner by higher concentrations of the peptide as was the activity of the MCH1-14 fragment analog. The MCH activities of the MCH5-17 and MCH5-14 analogs were not compromised by even the highest concentrations of the peptides employed. The MSH-like activity of MCH appears to relate to the N-terminus of the peptide whereas MCH activity is more a function of the C-terminus of the hormone. Self-antagonism of MCH at high concentrations appears to relate to the N-terminal tetrapeptide, which is responsible for the intrinsic MSH-like activity of the hormone.

Animals↗
PubMed1987

Topical application of a melanotropic peptide induces systemic follicular melanogenesis.

We determined the relative effectiveness of alpha-MSH and a highly potent melanotropin analogue, [Nle4, D-Phe] - alpha-MSH, in stimulating a shift from pheomelanogenesis to eumelanogenesis within hair bulbs of mice. The analogue proved to be at least a hundred times more effective than the native hormone when injected subcutaneously. The two melanotropins were then incorporated into an ointment base and topically applied to a shaved area of the skin on the back of a yellow strain of mice (C57BL/6JAY). Within 24-48 hours eumelanin production was visible within hair bulb melanocytes in both treated and untreated areas of animals. The presence of melanized organelles (eumelanosomes) within melanocytes was confirmed by electron microscopy. These results document the delivery of a peptide hormone through the skin and into the systemic circulation. This is the first demonstration of the delivery of a peptide hormone by percutaneous absorption and may provide a model for a similar route of delivery of other peptide hormones. The hormone analogue has also been delivered across human skin in vitro. Delivery of a melanotropin by a transdermal route may prove to be clinically useful in the treatment of some integumental hypopigmentary disorders in humans.

Administration, Topical↗
PubMed1987

The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism.

Treatment of intact hepatocytes with glucagon, TH-glucagon [( 1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/loss of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in a washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein Gs, and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.

Adenylyl Cyclases↗