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V J Hruby

Publications and source records attributed to V J Hruby.

At least 361 records · Page 20Linked to original sources
PubMed1986

Actions of melanotropins on mouse melanoma cell growth in vitro.

Melanotropins induce melanogenesis in mouse Cloudman S91 melanoma cells by stimulating the activity of tyrosinase. In monolayer culture, alpha-melanocyte-stimulating hormone and the superpotent analogue 4-norleucine, 7-D-phenylalanine-alpha-melanocyte, which had prolonged effects on tyrosinase activity, did not inhibit the proliferation of melanoma cells even at concentrations that elicited maximal tyrosinase stimulation. In soft agar the melanotropins stimulated the formation of melanized colonies and increased the cloning and proliferative potentials of melanoma cells. Both melanotropins increased the number of small (42-104 microns in diameter) colonies at initial plating densities ranging from 625 to 7,500 cells/dish. The number of larger (greater than 104 microns in diameter) colonies was also increased except at densities 5,000 cells or more/dish, wherein the proliferative capacity was inhibited; yet the cloning efficiency was still increased. Therefore, in bilayer soft agar cultures, melanotropins stimulate the growth of the clonogenic S91 melanoma cell population under conditions that allow for optimal expression of the cloning and proliferative potentials of these cells.

Animals↗
PubMed1986

Light microscopic autoradiographic localization of delta opioid receptors in the rat brain using a highly selective bis-penicillamine cyclic enkephalin analog.

Light microscopic autoradiography was used to visualize the neuroanatomical distribution of rat brain delta opioid receptors. Slide-mounted sections of rat brain were labeled with [3H]-[2-D-penicillamine, 5-D-penicillamine]enkephalin([3H]DPDPE), a highly selective delta opioid agonist. Saturation isotherms of [3H]DPDPE binding to thaw-mounted brain slices gave a maximal number of binding sites of 79.9 fmol/mg of protein and an apparent dissociation constant (Kd) of 6.3 nM. DPDPE and met-enkephalin inhibited [3H]DPDPE binding with high affinity (lC50 values of 6.3 and 13.8 nM, respectively). Putative mu opioid receptor selective ligands such as morphine sulfate, Tyr-D-Ala-Gly-NMePhe-Gyl-ol and [N-MePhe3, D-Pro4]morphiceptin (PL017) were less potent inhibitors of [3H]DPDPE binding. The rat brain areas containing the highest densities of receptors were the claustrum, basolateral amygdaloid nucleus, the caudate-putamen and nucleus accumbens, the external plexiform layer of the olfactory bulb and the olfactory tubercle. Moderate receptor density was characteristic of the hippocampal formation in which grains were seen over the molecular layer of the dentate gyrus and stratum oriens (CA1), and of the different layers of cerebral cortex. Generally, low density of binding was found over the thalamus and the septal nuclei. Low specific binding was also seen in the cerebellum, medulla oblongata and in the dorsal horn of the spinal cord. There was little specific [3H]DPDPE binding over the white matter areas.

Animals↗
PubMed1986

Comparative biological activities of potent analogues of alpha-melanotropin. Effect of nonaromatic and para substituted aromatic amino acids at position 7.

Several alpha-melanotropin (alpha-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO2), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle4]-alpha-MSH4-11-NH2 fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle4, Ala7]-alpha-MSH4-11-NH2 was more potent than Ac-[Nle4, D-Ala7]-alpha-MSH4-11-NH2 in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of alpha-MSH, to biological potency of the hormone.

Amino Acid Sequence↗
PubMed1986

Design of oxytocin antagonists with prolonged action: potential tocolytic agents for the treatment of preterm labor.

In our continuing effort to produce more potent and specific oxytocin (OT) antagonists that may have value as tocolytic agents, we have synthesized a number of new OT antagonists. Our previous studies have shown that rigid conformational structure and restricted dynamic properties are associated with antagonistic activity of the [1-penicillamine]OT [( Pen1]OT) analogs. We therefore synthesized a series of structural analogs of [Pen1,] OT; [Pen1,Thr4]-OT and [Pen1,Phe2,Thr4]OT with greater restricted conformational features. They are [Pen1,delta 3,4-Pro7]OT; [Pen1,Thr4,delta 3,4-Pro7]OT; [Pen1,Phe2,Thr4,delta 3,4-Pro7]OT; [Pen1, Orn8]OT; [Pen1,Phe2,Thr4,delta 3,4,-Pro7,Orn8]OT; [Pen1, Tyr(OMethyl)2,-Thr4,Orn8]OT; [Pen1,Tyr(OEthyl)2,Thr4,Orn8]OT; [Pen1,Phe(Methyl)2,Thr4,Orn8]OT and [Pen1Phe(Ethyl)2,Thr4, Orn8]OT. As expected, all were found to be potent OT antagonists, with in vitro pA2 values ranging from 5.32 to 7.67. They were also effective OT antagonists in vivo in the term pregnant rats. Structural modifications in the above analogs produced various and interesting effects. Dehydroproline substitution for 7-proline in [Pen1]OT increased antagonistic potency, whereas in [Pen1,Thr4]OT and in [Pen1,Phe2,Thr4]OT decreased antagonistic potency. Most significantly, analogs with O-alkyl-Tyr2, Orn8 and p-alkyl-Phe2,Orn8 substitutions were found to have prolonged action both in the isolated rat uterus assays and in the term pregnant rats. Generally, substitution of the alkyl groups resulted in a reduction in anti-OT potency, and increasing the size of the alkyl substituent from a methylene group to an ethyl group diminished antagonistic potencies markedly.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkylation↗
PubMed1985

Active and inactive L-prolyl-L-leucyl glycinamide synthetic analogs in rat models of levodopa-treated Parkinson's disease.

The tripeptide, L-prolyl-L-leucyl-glycinamide (PLG) has been shown to facilitate dopaminergic mechanisms in the brain. In the present study, we evaluated the interaction of PLG and its synthetic analogs with levodopa in two animal models of Parkinson's disease. In one experiment using rats with chronic unilateral lesions of the nigrostriatal dopamine pathway, PLG and Z-PLG potentiated the contraversive rotation elicited by levodopa with carbidopa (L/C). In a second experiment using reserpinized rats, PLG, Z-PLG and cyclo-LG potentiated L/C reversal of hypokinesia. Further studies of the PLG analogs, Z-PLG and cyclo-LG as adjunctive drugs with levodopa in the treatment of parkinsonism are warranted.

Animals↗
PubMed1985

Conformationally restricted cyclic analogues of substance P: insight into the receptor binding process.

Three new cyclic substance P analogues were prepared to examine the possible role of a pseudocyclic turn structure for receptor recognition. In the guinea pig isolated ileum [Cys5, Cys11]-SP5-11-NH2 and [Cys6, Cys11]-SP5-11-NH2 were inactive at concentrations up to 100 microM, while [Cys5, Cys6, Nle11]-SP was a weak agonist. The order of relative affinities on the rat brain radioreceptor assay was as follows: [Cys5, Cys6, Nle11]-SP greater than [Cys5, Cys11]-SP5-11-NH2 greater than [Cys6, Cys11]-SP5-11-NH2. We interpret these results to indicate that a pseudocyclic structure of the 5-11 sequence may not be an important factor involved in the receptor recognition of substance P.

Animals↗
PubMed1985

Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores.

The in vitro biological actions of synthetic chum salmon melanin concentrating hormone (MCH) on melanophores of the blue damselfish (a teleost), Chrysiptera cyanea, were studied. This cyclic heptadecapeptide stimulated melanosome (melanin granule) aggregation (centripetal migration) within melanophores at a threshold concentration of about 10(-10) M. The action of this putative hormone was not blocked by alpha- or beta-adrenoceptor antagonists. It was concluded that the effects of MCH were direct and were not mediated indirectly through the actions of adrenergic neurotransmitters released from nerve terminals. Further evidence for this view comes from the observation that, unlike the case of neurotransmitter release, melanosome aggregation in response to MCH proceeded in the absence of calcium. The possible role of MCH in the control of color change of teleost fishes is discussed.

Animals↗
PubMed1985

Synthesis and biological evaluation of the superagonist [N alpha-chlorotriazinylaminofluorescein-Ser1,Nle4,D-Phe7]-al pha-MSH.

The fluorescein-labeled melanotropin [N alpha-chlorotriazinylaminofluorescein-Ser1,Nle4,D-Phe 7]-alpha-MSH, was prepared by solid-phase techniques of peptide synthesis. The biological actions of this analogue were determined in several melanocyte bioassays and were compared with the parent peptide [Nle4,D-Phe7]-alpha-MSH and the native hormone alpha-MSH. The fluorescein compound was a superpotent agonist with approximately 10 times more activity than alpha-MSH in both the frog and the lizard skin bioassays. Murine S91 melanoma cells assayed in vitro (tyrosinase bioassay) were as responsive to the fluorescein analogue as to alpha-MSH. The analogue exhibited ultraprolonged biological activity and the biological activities were unaffected by treatment of the analogue with alpha-chymotrypsin. The fluorescein-labeled melanotropin should prove useful for melanotropin receptor characterization.

Animals↗
PubMed1985

Studies in vitro with ICI 174,864, [D-Pen2, D-Pen5]-enkephalin (DPDPE) and [D-Ala2, NMePhe4, Gly-ol]-enkephalin (DAGO).

The interactions of a proposed, selective delta receptor antagonist (ICI 174,864) and selective agonists at mu and delta receptors, [D-Ala2, NMePhe4, Gly-ol]-enkephalin (DAGO) and [D-Pen2, D-Pen5]-enkephalin (DPDPE), respectively, have been studied using the electrically-stimulated mouse isolated vas deferens (MVD) and the guinea-pig isolated ileum (GPI). Incubation of increasing concentrations of ICI 174,864 (10,30,100 and 300 nM) produced a dose-related and parallel rightward displacement of the DPDPE dose-response curve in the MVD. In contrast, ICI 174,864 (300-3000 nM) failed to affect the DAGO dose-response curve in the same tissue. Analysis of the DPDPE-ICI 174,864 interaction in the MVD using the pA2 method revealed a Schild plot slope of -0.68 suggesting the involvement of more than one population of receptors. ICI 174,864 (300 nM) failed to antagonize DPDPE in the GPI at doses up to 30 microM. These results suggest that (a) ICI 174,864 acts as a selective delta antagonist in the MVD; (b) DPDPE interacts with mu receptors in the MVD but only at very high concentrations, and (c) delta receptors appear not to be of functional importance in the GPI.

Animals↗
PubMed1985

Somatostatin analogs with affinity for opiate receptors in rat brain binding assay.

The somatostatin analogs D-Phe-Cys-D-Trp-Lys-Thr-Cys-Thr and the corresponding penicillamine compounds have been prepared and tested for their ability to displace [3H]naloxone and [3H] [D-Ala2, D-Leu5]enkephalin from rat brain receptors. While somatostatin and the cystine containing peptide displayed little or no preference for either receptor system, the substitution of penicillamine at position two or seven resulted in analogs that displayed opposite receptor selectivity. The substitution of tyrosine for phenylalanine at position three resulted in a large increase in opiate receptor affinity which may be related to the known requirement for a phenolic hydroxyl moiety in the rigid opiate and enkephalin systems. Conformational properties of these analogs were also examined and related to their affinity for opiate and somatostatin receptors in the rat brain.

Animals↗
PubMed1985

[Nle4, D-Phe7]-alpha-MSH: a superpotent melanotropin with prolonged action on vertebrate chromatophores.

The in vitro and in vivo responses of integumental chromatophores to alpha-MSH and a related analogue, [Nle4, D-Phe7] -alpha-MSH, were studied in a number of vertebrate species: the teleost, Lebistes reticulatus; the amphibians, Rana pipiens, R. catesbeiana, Xenopus laevis, Bufo alvarius, and B. cognatus; the lizard, Anolis carolinensis; the rattlesnake, Crotalus atrox. The alpha-melanotropin analogue was a superpotent agonist in the in vitro frog (R. pipiens, R. catesbeiana) and lizard (A. carolinensis) skin bioassays. In all species studied, the analogue exhibited ultraprolonged melanotropic activity, both in vitro and in vivo. This melanotropin and related analogues should prove useful in the study of numerous physiological processes, particularly when prolonged melanotropic activity is desired.

Animals↗
PubMed1985

Stimulation of S91 melanoma tyrosinase activity by superpotent alpha-melanotropins.

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin), [Nle4,D-Phe7]-alpha-MSH and related fragment analogues, Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 and Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, were studied for their ability to stimulate tyrosinase activity in Cloudman S91 mouse melanoma cells in tissue culture. All of the melanotropins stimulated tyrosinase activity in a dose-dependent manner. [Nle4,D-Phe7]-alpha-MSH was about 100 times more active than alpha-MSH as determined from the minimal effective dose (MED) required to activate the enzyme above control (basal) levels. The MED of this analogue to significantly stimulate tyrosinase activity at 24, 48 and 72 h of incubation was 10(-11) M whereas the MED of alpha-MSH was 10(-9) M at each of these times. The maximum tyrosinase activity achieved from the time of initial incubation in the presence of [Nle4,D-Phe7]-alpha-MSH was approximately 3-, 5- and 6-fold greater than control levels at 24, 48 and 72 h, respectively. The 2 [Nle4,D-Phe7]-substituted fragment analogues were at least as active as the tridecapeptide analogue and therefore at least 100-fold more active than alpha-MSH in stimulating enzyme activity. These [Nle4,D-Phe7]-substituted analogues were more active in the melanoma tyrosinase assay than in the melanoma adenylate cyclase assay or other normal melanocyte (frog and lizard skin) bioassays.

Amino Acid Sequence↗
PubMed1985

Cyclic melanotropins. 9. 7-D-Phenylalanine analogues of the active-site sequence.

The cyclic melanotropin Ac-Ser1-Tyr2-Ser3-Cys4-Glu5-His6-Phe7-Arg8 -Trp9-Cys10-Lys11-Pro12-Val13-NH is a highly potent agonist as determined in several melanocyte bioassays. In linear melanotropins, a D-Phe7 substitution leads to increased potency and often prolonged biological activity. In order to determine if this substitution would have the same effect in cyclic melanotropins, we have prepared a series of these analogues. The D-Phe7-substituted cyclic melanotropins Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 and Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-11-NH2 were both more potent than their cyclic L-Phe7-containing counterparts in either the frog or lizard skin bioassay by more than a factor of 10. Neither peptide, however, exhibited prolongation of biological activity in either assay. Substitution of D-Phe7 into the cyclic 4-12 and 4-13 sequences led to a slight or no increase in potency in both assays relative to the L-Phe7 counterparts, but the activity of the melanotropins was ultraprolonged in each assay. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-12-NH2 was about equipotent to Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-13-NH2, again demonstrating, as with certain linear and cyclic L-Phe7-containing melanotropins, that the C-terminal amino acid valine is not required for biological activity or for superpotency. Similar to the linear D-Phe7 analogues that possessed ultraprolonged melanotropic activity, the 4-12 and 4-13 cyclic D-Phe7 analogues also displayed the phenomenon of superagonism, which is a time-dependent increase in efficacy over that produced by an equipotent concentration of the native hormone. Cyclization of certain linear melanotropins resulted in analogues with increased resistance to biological degradation by serum enzymes or purified proteolytic enzymes. Further, incorporation of a D-Phe7 into in the cyclic analogues led to melanotropins that were totally resistant to enzymatic inactivation by trypsin.

Animals↗
PubMed1985

Conformationally restricted analogs of somatostatin with high mu-opiate receptor specificity.

A series of cyclic, conformationally restricted analogs of somatostatin have been prepared and tested for their ability to inhibit the binding of [3H]naloxone and [D-Ala2, D-Leu5] [3H]enkephalin to rat brain membranes. The most potent analog, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 where Pen is penicillamine in [D-Phe5, Cys6, Tyr7, D-Trp8, Pen11]somatostatin-(5-12)-octapeptide amide, exhibited high affinity for mu-opiate receptors (IC50 value of [3H]naloxone = 3.5 nM), being 7800 times more potent than somatostatin. The cyclic octapeptide also displayed high mu-opiate receptor selectivity with an IC50 [( D-Ala2,D-Leu5]enkephalin)/IC50 (naloxone) ratio of 271. The high affinity and selectivity of the somatostatin analog for mu-opiate receptors may be of use in examining the physiological role(s) of the mu-opiate receptor.

Animals↗
PubMed1985

Characterization of [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin binding to delta opiate receptors in the rat brain and neuroblastoma--glioma hybrid cell line (NG 108-15).

Specific binding properties of the tritium-labeled delta opiate receptor agonist [3H][2-D-penicillamine, 5-D-penicillamine]enkephalin [( 3H][D-Pen2, D-Pen5]enkephalin) were characterized in the rat brain and in a mouse neuroblastoma-rat glioma hybrid cell line (NG 108-15). Saturation isotherms of [3H][D-Pen2, D-Pen5]enkephalin binding to rat brain and NG 108-15 membranes gave apparent Kd values of 1-6 nM. These values are in good agreement with the Kd value obtained from the kinetic studies. The Bmax value in NG 108-15 membranes was 235.3 fmol/mg of protein. An apparent regional distribution of [3H][D-Pen2, D-Pen5]enkephalin binding was observed in the rat brain. A number of enkephalin analogues inhibited [3H][D-Pen2, D-Pen5]enkephalin binding with high affinity (IC50 values of 0.5-5.0 nM) in both NG 108-15 and rat brain membranes. However, putative mu receptor-selective ligands such as morphine, [D-Ala2, MePhe4, Gly5-ol]enkephalin, [MePhe3, D-Pro4]morphiceptin, and naloxone were less effective inhibitors of [3H][D-Pen2, D-Pen5]enkephalin binding in both systems tested. These data suggest that [3H][D-Pen2, D-Pen5]enkephalin is a potent and selective ligand for the delta opioid receptor.

Animals↗