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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

Long-term and residual melanotropin-stimulated tyrosinase activity in S91 melanoma cells is density dependent.

Cell density is a factor that affects the capacity of Cloudman S91 melanoma cells to respond to melanotropins in monolayer culture. Continuous exposure of melanoma cells to alpha-melanotropin or its potent analog [Nle4, D-Phe7]-alpha-MSH, resulted in maximal stimulation of tyrosinase after 2 d of treatment, but the magnitude of stimulation decreased thereafter despite the continued presence of the melanotropins. However, when melanoma cells continually exposed to melanotropins were subcultured to an initial low cell density and maintained in contact with alpha-MSH or [Nle4, D-Phe7]-alpha-MSH (long-term culture), tyrosinase activity was rapidly restored and greatly enhanced. Also, when cells were seeded at initial densities ranging from 0.2 to 3.2 X 10(6) cells/flask, and exposed for 24 h to 10(-7) M alpha-MSH, only the cultures seeded at low densities (0.2 and 0.4 X 10(6) cells/flask) exhibited maximal tyrosinase activity during the 24 h exposure to the melanotropins. Therefore, tyrosinase activity was primarily affected by cell density rather than by the duration of time the cells were in culture or by continuous exposure to melanotropin. Other flasks of various cell densities were treated with 10(-7) M alpha-MSH or [Nle4, D-Phe7]-alpha-MSH for 24 h, followed by removal of the melanotropins from the culture medium. The magnitude and duration of the residual stimulation of melanoma tyrosinase activity by melanotropins were also found to be dependent on the initial cell density. These results reveal that there is a limited range of optimal cell densities at which melanoma cells can respond to melanotropins and express increased tyrosinase activity.

Animals↗
PubMed1986

Action of melanin-concentrating hormone (MCH) on teleost chromatophores.

The in vitro effects of synthetic salmon melanin-concentrating hormone (MCH) on chromatophores of four teleost species were studied. In the erythrophores of the platyfish (Xiphophorus maculatus) and the swordtail (Xiphophorus helleri), and in the xanthophores and amelanotic melanophores of the medaka (Oryzias latipes), pigment aggregation took place in response to MCH even in the absence of Ca2+. In contrast to this, the leucophores of the medaka responded to MCH by the pigment dispersion but only when Ca2+ was present. The motile iridophores of the blue damselfish (Chrysiptera cyanea), which play a predominant role in coloration and its changes, were not affected by the hormone. Pharmacological studies employing various blocking agents suggest that the pigment-aggregating action of MCH is probably mediated through specific receptors possessed by the erythrophores, xanthophores, or amelanotic melanophores, while the pigment-dispersing action on the leucophores might be revealed through the receptors for melanophore-stimulating hormone (MSH).

Animals↗
PubMed1986

Is oxytocin-induced grooming mediated by uterine-like receptors?

In this study, we examined whether the mechanisms mediating the induction of grooming behavior by oxytocin (OT) is similar to mechanisms mediating the effects of OT on uterine contractility. Sprague-Dawley strain female rats were injected intracerebroventricularly (ICV) with OT or OT analogues and then were observed for grooming behaviors 25 minutes later for 30 minutes. The uterotonic analogue deamino-OT injected ICV at equimolar doses to 1 microgram OT significantly elevated grooming scores although less than did OT. Other agonist analogues were not effective in inducing an increase in grooming behavior. The simultaneous ICV injection of the analogue [Pen1, Phe2, Thr4, delta 3, 4Pro7, Orn8]-OT, which blocked the effects of OT on uterine contractility, also blocked the effect of OT on grooming behavior. Injection of the same dose of this antagonist analogue did not effect the increased grooming behavior after AVP injection. Pretreatment with 5 mg/kg of the prostaglandin synthetase inhibitor indomethacin significantly inhibited OT-induced grooming. We have concluded from these data that the mechanism underlying the effect of OT on grooming is similar to its effects on uterine contractility in some respects. However, observations that the OT antagonist analogue blocked OT- but not AVP-induced grooming may suggest that more than one receptor or mechanism exists by which nonapeptides initiate excessive grooming.

Animals↗
PubMed1986

Design and synthesis of conformationally constrained somatostatin analogues with high potency and specificity for mu opioid receptors.

A series of cyclic, conformationally constrained peptides related to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the mu opioid receptor. The following new peptides were prepared and tested for their mu opioid receptor potency and selectively in rat brain binding assays: D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (2, CTOP); D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (3, CTAP); D-Phe-Cys-Tyr-D-Trp-Nle-Thr-Pen-Thr-NH2 (4); D-Phe-Cys-Tyr-D-Trp-Lys-Val-Pen-Thr-NH2 (5); D-Phe-Cys-Tyr-D-Trp-Lys-Gly-Pen-Thr-NH2 (6); D-Phe-Cys-Tyr-Trp-Lys-Thr-Pen-Thr-NH2 (7); D-Tyr-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH (8); D-PhGly-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (9); and D-PhGly-Pen-Phe-D-Trp-Lys-Thr-Cys-Thr-OH (10). The most selective peptide, 2 (CTOP), displayed both high affinity (IC50 = 3.5 nM) and exceptional selectivity (IC50 delta/IC50 mu = 4,000) for mu opioid receptors. Furthermore, 2 exhibited very low affinity for somatostatin receptors in the rat brain (IC50 greater than 24,000 nM), with an IC50 somatostatin/IC50 mu receptor selectivity of 8,750. These conformationally constrained cyclic peptides should provide new insight into the structural and conformational requirements for the mu opioid receptor and the physiological role of this receptor.

Animals↗
PubMed1986

H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2: a potent and selective antagonist opioid receptors.

H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) exhibited high affinity (IC50 = 2.80 nM) in displacing [3H]naloxone binding (nH = 0.89 +/- 0.1) and showed an exceptional selectivity for mu opioid receptors with an IC50(DPDPE)/IC50(naloxone) ratio of 4,840, while it displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM) in rat brain binding assays. [3H]CTOP was recently custom synthesized (spec. act.: 84 Ci/mmol) and evaluated for its in vitro binding properties towards the mu opioid receptors in rat brain membrane preparations. Association and dissociation of [3H]CTOP binding to mu opioid receptors were rapid at 25 degrees C with a kinetic Kd value of 0.67 nM. Saturation experiments gave apparent Kd value of 1.11 nM and Bmax value of 136 +/- 13 fmol/mg prot at 25 degrees C. Specific [3H]CTOP binding was inhibited by a number of different opioid and opiate ligands. Among them, putative mu opioid receptor-specific ligands, such as naloxone, naltrexone and CTOP inhibited the binding with high affinity, while delta opioid receptor-specific compounds or non-opioid drugs inhibited specific [3H]CTOP binding with low affinity or they were ineffective.

Animals↗
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↗