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V J Hruby

Publications and source records attributed to V J Hruby.

At least 181 records · Page 10Linked to original sources
PubMed1988

Perifused precision-cut liver slice system for the study of hormone-regulated hepatic glucose metabolism.

A nonrecirculatory perfusion system for precision-cut rat liver slices has been developed and utilized for investigating hormone-regulated hepatic glucose metabolism. In this system, slices are cultured in a highly controlled environment and exhibit excellent retention of viability as judged by their maintenance of intracellular potassium and glycogen contents. Using this system, the complex physiological phenomenon of hormone-regulated glycogenolysis was investigated at both extra- and intracellular sites. Specifically, the sensitive responses of intracellular cyclic AMP (cAMP) production, activation of cyclic AMP-dependent protein kinase, and production of glucose upon glucagon stimulation have been measured. The maximal responses observed for these parameters were either equal to or greater than those previously reported for either isolated hepatocytes or perfused livers, demonstrating the sensitivity of this technique. Upon dose-response examination of glucagon challenge, it was observed that high doses of glucagon (greater than 16 nM) stimulate glucose production by activating the cAMP-second messenger cascade. In contrast, low doses (less than 4 nM) stimulate this process without production of intracellular cAMP or activation of cAMP-dependent protein kinase, suggesting the operation of cAMP-independent messenger. Since this system permits measurements of parameters common to many cellular processes, this methodology is suitable for addressing both pharmacological and toxicological questions.

Animals↗
PubMed1988

Ventral tegmental oxytocin induces grooming.

Bilateral microinjection of oxytocin (OXY) into the ventral tegmental area (VTA) of rat brain produced a significant increase in grooming behaviors at doses from 100 pg to 400 ng. Sites in the caudal region of the VTA were sensitive to lower doses of OXY than sites in the rostral region of the VTA. The time course of action of OXY in the grooming paradigm indicated onset beginning immediately after injection, and termination at 60-75 minutes after injection. Comparison of OXY-induced grooming in male, female, and ovariectomized, estrogen-treated female rats showed no differences in potency for OXY among these groups, suggesting that the grooming effects of OXY are not regulated by sex steroids. Analysis of locomotor activity in rats microinjected with OXY 200 ng bilaterally into the caudal VTA revealed that OXY had no effect on ambulatory locomotion, suggesting that this peptide may activate neurons within the VTA which mediate grooming but not locomotion. The OXY receptor antagonist, [Pen1, pMePhe2, Thr4, Orn8]-OT, blocked OXY-induced grooming when both were simultaneously microinjected into the VTA. The dopamine D-2 receptor antagonist, haloperidol, and the D-1 receptor antagonist, SCH 23390, when microinjected into the VTA five minutes before microinjection of OXY into the VTA, did not block OXY-induced grooming, suggesting that OXY is not working through a dopamine autoreceptor on the VTA neurons. Systemic pretreatment with haloperidol and SCH 23390 effectively blocked grooming induced by OXY in the VTA, suggesting that OXY is directly stimulating OXY receptors on VTA neurons to release dopamine at postsynaptic sites regulating grooming behaviors.

Animals↗
PubMed1988

The effects of glucagon and TH-glucagon on steroid metabolism in isolated rat hepatocytes.

Glucagon decreases the activity of steroid-metabolising enzymes in isolated rat liver cells at physiological concentrations. Higher concentrations are less effective. TH-glucagon (1-N-alpha-trinitrophenylhistidine-12-homoarginine-glucagon) also reduces enzyme activity but does not lose activity at higher concentrations. The effects of the two hormones mimic closely their reported effects on phosphatidylinositol-4,5-bisphosphate breakdown. It is, thus, likely that the effect of glucagon on steroid metabolism is mediated via breakdown of this phospholipid. The calcium ionophore, A23187, had no effect on steroid metabolism whereas the phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) mimicked the effect of glucagon, showing that activation of protein kinase C but not Ca2+ mobilization may be involved in glucagon's action on hepatic steroid metabolism.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1988

Design and synthesis of somatostatin analogues with topographical properties that lead to highly potent and specific mu opioid receptor antagonists with greatly reduced binding at somatostatin receptors.

A series of conformationally restricted, cyclic octapeptides containing a conformationally stable tetrapeptide sequence related to somatostatin, -Tyr-D-Trp-Lys-Thr-, as a template, were designed and synthesized with the goal of developing highly potent and selective mu opioid antagonists with minimal or no somatostatin-like activity. Three distinct structures of the peptide became targets of chemical modifications and constraints; the N- and C-terminal amino acids and the cyclic 20-membered ring moiety. Based on the conformational analysis of active and inactive analogues of the parent peptide D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Thr6-Pen7+ ++-Thr8-NH2, CTP (Kazmierski, W.; Hruby, V. J. Tetrahedron 1988, 44, 697-710), we designed analogues to include the tetrahydroisoquinolinecarboxylate (Tic) moiety as the N-terminal amino acid instead of D-Phe, since Tic can exist only as a gauche (-) or a gauche (+) conformer. In this series, the following peptides were synthesized and pharmacologically evaluated: D-Tic-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (TCTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (TCTOP), and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (TCTAP). In rat brain membrane opioid radioligand binding assays, all three peptides displayed high affinity for mu opioid receptors (IC50 = 1.2, 1.4, 1.2 nM, respectively), and exceptional mu vs delta opioid receptor selectivity: 7770, 11,396, and 1060, respectively. TCTOP and TCTAP also possess exceptional mu vs somatostatin receptor selectivity: 14,574 and 28,613, respectively. In the peripheral in vitro GPI bioassay, TCTP, TCTOP, and TCTAP were highly effective antagonists of the potent mu opioid receptor agonist PL017, with pA2 = 8.69 for TCTAP, 8.10 for TCTP, and 7.38 for TCTOP. Our results show that a 10-fold higher affinity and selectivity for mu opioid receptors (in both central and peripheral studies) over delta and somatostatin receptor was gained as a result of the D-Tic1 substitution. These three peptides, TCTP, TCTOP, and TCTAP, are the most potent and selective mu opioid antagonists known. CTP has been shown to possess prolonged biological action, much longer than that of naloxone. This renders these analogues potentially useful ligands for investigating the physiological functions of the mu opioid receptor. Analogues of TCTP in which the 20-membered disulfide ring was contracted by deletion of D-Trp4, and/or Lys5, and/or Thr6 led to compounds with greatly reduced potency at the mu opioid receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acids↗
PubMed1988

Melanin concentrating hormone analogues: contraction of the cyclic structure. 1. Agonist activity.

Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which is synthesized in the hypothalamus and secreted by the neurohypophysis of teleost fishes. This hormone exhibits both MCH-like as well as alpha-MSH (alpha-melanocyte stimulating hormone) like activity. We have examined the role of the disulfide bond for the two contrasting melanotropic activities of MCH. Nine analogues of the parent peptide were synthesized and characterized for biological activity. The disulfide ring was contracted from the 5-14 to the 7-14, 8-14, and 10-14 residues with concomitant substitution of alanine for Cys at position 5 in each of the heptadecapeptides. Similar substitutions were made in a series of MCH analogues. In addition, the following cyclic peptides also were synthesized: [Cys7]MCH, [Cys8]MCH, and [Cys10]MCH. The fish-skin bioassay is sensitive to MCH at a concentration of 10(-12) M. All ring-contracted analogues were inactive at 10(-6) M or lower concentrations; less than 1/1,000,000 compared to MCH (1.0) except [Ala5,Cys8]MCH (0.0008; 1/1250), [Cys10]MCH (0.000 09; 1/10,000), and [Cys8]MCH (0.000 001; 1/1,000,000). In the frog-skin bioassay, [Ala5,Cys10]MCH, although lacking MCH-like activity in the fish-skin bioassay, was equipotent to MCH in its alpha-MSH-like component of activity. Most other analogues were either inactive or much less active than MCH in stimulating melanosome dispersion. These results demonstrate that the disulfide bond between positions 5 and 14 is essential for the MCH-like activity since contraction of the ring generally leads to inactive peptides. Contraction of the disulfide bridge does not, however, have as great an effect on the MSH-like activity of MCH.

Animals↗
PubMed1988

Stimulation and antagonism of opioid delta-receptors produce opposite effects on active avoidance conditioning in mice.

The effects of opioid delta-receptor activation on conditioning in a one-way active avoidance paradigm were investigated in mice. Peripheral administration of 30 and 100 micrograms/kg of [Leu5]enkephalin (LE) and 11.6 micrograms/kg of [D-Pen2,D-Pen5]enkephalin (DPDPE), a synthetic enkephalin analog with high delta-selectivity, impaired acquisition of avoidance responding. The dose response functions for both peptides were U-shaped. Deficits in responding were also present 24 hours after training, which suggests that the impaired performance observed during training was not an impairment in the ability of the mice to perform. Also, neither LE nor DPDPE decreased footshock sensitivity or open-field activity, which rules out analgesia and decreased activity levels as likely explanations for the deficits in conditioning. Finally, antagonizing endogenous ligands with the delta-selective antagonist ICI 154,129 enhanced acquisition, an effect opposite that produced by LE and DPDPE. The results suggest that activation of delta-receptors is normally involved in the modulation of active avoidance learning.

Animals↗
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↗