PubMed Health⌕ Search

Biomedical subjects

V J Hruby

Publications and source records attributed to V J Hruby.

At least 91 records · Page 5Linked to original sources
PubMed1989

Design of potent linear alpha-melanotropin 4-10 analogues modified in positions 5 and 10.

alpha-Melanocyte stimulating hormone (alpha-MSH) is a linear tridecapeptide (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) that has diverse physiological functions in addition to its reversible darkening of amphibian skins by stimulating melanosome dispersion within melanophores. On the basis of theoretical and experimental results from our laboratory and others, we have designed a group of 1-13, 4-13, and especially 4-10 analogues related to the superpotent analogue [Nle4,D-Phe7]alpha-MSH in which the Glu5 has been replaced with Asp5, and the Gly10 has been replaced with Lys10 and other basic amino acid residues in the 4-10 analogues, and in which Gly10 and Lys11 were interchanged in the longer peptide analogues. In the 1-13 and 4-13 series the Lys10, Gly11 analogues generally retained superpotency for the D-Phe7-containing analogues. Most interestingly, synthesis of Ac-[Nle4,Xxx5,Yyy7,Zzz10]alpha-MSH4-10-NH2 analogues where Xxx = Asp or Glu, Yyy = Phe or D-Phe, and Zzz = basic amino acids (Lys, Orn, alpha,gamma-diaminobutyric acid (Dab), and alpha,beta-diaminopropionic acid (Dpr] provided melanotropins with potencies up to 10 times that of the native hormone in stimulating frog (Rana pipiens) skin darkening and 8-50 times more potent than alpha-MSH in stimulating lizard (Anolis carolinensis) skin melanophores in vitro. To our knowledge, Ac-[Nle4,Asp5,D-Phe7,Dab10]alpha-MSH4-10-NH2, the most potent analogue, is the most potent melanotropin obtained thus far for the Anolis assay system. These results provide new insights into the structural and conformational requirements for biological potency of alpha-MSH and the differential structural and conformational requirements of alpha-MSH and its analogues at two different types of pigment cell receptors.

Animals↗
PubMed1989

Synthesis of highly mu and delta opioid receptor selective peptides containing a photoaffinity group.

A series of cyclic, conformationally constrained photolabile peptides related to the enkephalins and to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the delta and mu opioid receptors. The following new peptides were prepared and tested for their delta opioid receptor potency and selectivity in the guinea pig ileum assay, the mouse vas deferens assay, and the rat brain binding assay: H-Tyr-D-Pen-Gly-p-NH2Phe-D-Pen-OH (1, [p-NH2Phe4]DPDPE) and H-Tyr-D-Pen-Gly-p-N3Phe-D-Pen-OH (2, [p-N3Phe4]-DPDPE). The following new peptides were prepared and tested for their mu opioid receptor potency and selectivity in the same assays: H-D-Phe-Cys-p-NH2Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (3, [p-NH2Phe3]CTP) and D-Phe-Cys-p-N3Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (4, [p-N3Phe3]CTP). The delta selective photoaffinity peptide 2 displayed both high affinity (IC50 = 9.5 nM) and good selectivity (IC50 mu/IC50 delta = 1053) as an agonist at delta opioid receptors in bioassays, and 2 also displayed moderate affinity (33 nM) and excellent selectivity (IC50 mu/IC50 delta = 110) for rat brain delta opioid receptors. The mu selective photoaffinity peptide 4 displayed very weak affinity (8% contraction at 300 nM) at mu opioid receptors in bioassays, but good affinity (IC50 = 48.6 nM) and excellent selectivity (IC50 delta/IC50 mu = 412) for the rat brain mu opioid receptors. These conformationally constrained cyclic photoaffinity peptides may be useful tools to investigate the pharmacology of delta and mu opioid receptors.

Affinity Labels↗
PubMed1989

Potent and prolonged acting cyclic lactam analogues of alpha-melanotropin: design based on molecular dynamics.

Utilizing results from previous structure-activity relationships and theoretical studies of alpha-melanotropin (alpha-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2) and its related superpotent analogues, Ac-[Nle4,D-Phe7]-alpha-MSH and Ac-[Cys4,Cys10]-alpha-MSH, we have designed a new class of alpha-MSH4-13 and alpha-MSH4-10 cyclic lactam fragment analogues of alpha-melanotropin. The cyclic peptides have the following general structures: Ac-[Nle4,Xxx5,D-Phe7,Yyy10,Gly11]-alpha-MSH4-13- NH2 and Ac-[Nle4,Xxx5,D-Phe7,Yyy10]-alpha-MSH4-10-NH2, where Xxx = Glu or Asp and Yyy = Lys, Orn, Dab, or Dpr. Formation of the lactam bridge between the side-chain groups Xxx and Yyy was performed either in solution or on a solid-phase support. Seven cyclic peptides were prepared and bioassayed for their melanotropic potency by using standard frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. Relative to alpha-MSH (relative potency = 1), the potencies of the cyclic peptides in the lizard skin bioassay were as follows: alpha-MSH (1); Ac-[Nle4,Glu5,D-Phe7,Lys10,Gly11]-alpha-MSH4-13- NH2 (6); Ac-[Nle4,Asp5,D-Phe7,Lys10,Gly11]-alpha-MSH4-13- NH2 (100); Ac-[Nle4,Glu5,D-Phe7,Lys10]-alpha-MSH4-10-NH2 (9); Ac-[Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH4-10-NH2 (90); Ac-[Nle4,Asp5,D-Phe7,Orn10]-alpha-MSH4-10-NH2 (20); Ac-[Nle4,Asp5,D-Phe7,Dab10]-alpha-MSH4-10-NH2 (5); Ac-[Nle4,Asp5,D-Phe7,Dpr10]-alpha-MSH4-10-NH2 (5). Similar results were obtained in the frog skin bioassay, but the analogues were much less potent. Cyclic melanotropins with 23-membered rings exhibited 100-fold higher melanotropic potency than alpha-MSH with selectivity for the lizard melanocyte receptors over the frog melanocyte receptors. Increasing or decreasing the ring size of these cyclic melanotropins from 23 diminishes the biological potency of the resulting cyclic peptide. The 23- and 24-membered ring analogues showed prolonged (residual) biological activities in both biological assays, but the smaller ring systems (20, 21, 22) did not. These results provide new insights into the structural and conformational requirements of alpha-MSH and its analogues at two different types of pigment cell (melanocyte) receptors.

Amino Acid Sequence↗
PubMed1989

Influence of opioid peptides on learning and memory processes in the chick.

Several experiments were conducted to examine the effects of intracranial injection of opioid peptides and antagonists on learning and memory in the chick. Pretraining injection of [leu5]enkephalin and the selective delta receptor agonist [D-Pen2,L-Pen5]enkephalin (DPLPE) into the intermediate medial hyperstriatum ventrale (IMHV) produced impairment. ICI 174,864, a delta-selective antagonist, reversed the impairment produced by either [leu5]enkephalin or DPLE, results indicating that delta receptors may play a role in learning in the chick and suggesting that the impairment produced by [leu5]enkephalin is mediated through delta opioid receptors. beta-endorphin produced a naloxone-reversible impairment in performance, which suggests that this impairment is mediated by opioid receptors. Bilateral injection of beta-endorphin into the IMHV produced impairment, as did unilateral injection into the right, but not left, IMHV. Only bilateral injections into IMHV of [leu5]enkephalin were effective. These results suggest that the effects of beta-endorphin are centrally mediated whereas the effects of [leu5]enkephalin may be localized to other brain regions or are peripherally mediated. These initial results suggest that opioids are associated with learning and memory in the chick.

Animals↗
PubMed1989

Conformational analysis of enkephalin analogs containing a disulfide bond. Models for delta- and mu-receptor opioid agonists.

Conformational analysis of the cyclic opioids H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) and H-Tyr-D-Cys-Gly-Phe-D-Cys-OH (DCDCE) have been performed using the AMBER program. DPDPE is considerably more selective for delta-receptors than DCDCE. Using the RNGCFM program, a large number of ways were found to close the 14-membered disulfide-containing ring structure. However, intramolecular hydrogen bonds were only possible in gamma-turn and inverse gamma-turn conformations centered on the glycine residue which were associated with opposite chiralities of the disulfide bond. With the cyclic part of the molecules in either a gamma-turn or inverse gamma-turn, a systematic conformational analysis was performed on the tyrosine and phenylalanine sidechains. This showed that conformers with the tyrosine and phenylalanine phenyl rings in the vicinity of the disulfide bond were preferred due to attractive van der Waals forces. For DPDPE, however, this was only possible with a positive dihedral angle for the disulfide bond due to the presence of the beta-carbon methyls of Pen2. In contrast, these preferred conformers were possible with both chiralities of the disulfide bond in DCDCE. Conformational entropies and free energies were computed from the translational, rotational, and vibrational energy levels available to each conformer. The conformational entropies were found to vary significantly and to result in a re-ordering of the lowest energy minima. Based on these conformational differences in DPDPE and DCDCE and their differing pharmacological selectivities, tentative conformational preferences for delta- and mu-receptor opioid peptides are proposed.

Disulfides↗
PubMed1989

Designing molecules: specific peptides for specific receptors.

Peptides are the largest class of mediators of intercellular communication in the central nervous system. These molecules pose special problems in design for potential medical applications because of the high degree of flexibility, lack of high receptor selectivity, and ready biodegradation or clearance. The global and local use of conformational constraints has overcome these difficulties. Structure-biological activity relationships, molecular modeling, conformational analysis, conformational calculations, and molecular dynamics simulations can all be used to derive suitable lead structures and conformational models. Often, a single, constrained peptide analogue can be designed, which will have many of the desired biological and biophysical properties, and will serve as a template. Peptide analogues with high potency, exquisite receptor selectivity, and biological stability can be obtained. The approach is illustrated by the design of cyclic enkephalin-agonist analogues with exceptional delta-opioid-receptor selectivity, and of constrained somatostatin analogues that have become opioid peptides and possess potent opioid antagonist activities and exceptional selectivity for mu-opioid receptors.

Chemical Phenomena↗
PubMed1989

Antidiarrheal properties of supraspinal mu and delta and peripheral mu, delta and kappa opioid receptors: inhibition of diarrhea without constipation.

We evaluated the ability of mu [morphine, Tyr-Pro-N-MePhe-D-Pro-NH2 (PLO17)], delta (Tyr-D-Pen-Gly-Phe-D-Pen) (DPDPE) and kappa [U50,488H, (trans-3,4-dichloro-N-methyl-N-(2-(1-pyr-rolidinyl) cyclo-hexyl)benzeneacetamine)] opioid receptor selective agonists to inhibit diarrhea induced by castor oil (0.6 ml p.o.) in mice after supraspinal (i.c.v.) and peripheral (s.c.) administration. The antidiarrheal potency of each compound was compared to its analgesic and gastrointestinal antitransit potency when given by the same route of administration. When administered i.c.v., morphine, PLO17 and DPDPE inhibited diarrhea in a dose-related fashion. The mu agonists, morphine and PLO17, given i.c.v, inhibited diarrhea at doses much lower than those needed to produce analgesia or to inhibit gastrointestinal transit. DPDPE (i.c.v.) was equipotent in inhibiting diarrhea and in eliciting analgesia, but did not effect the rate of transit. U50,488H (i.c.v.) inhibited diarrhea only at extremely high doses which also caused profound postural-motor incapacitance. U50,488H given i.c.v. had no effect on transit at any dose. When given peripherally, morphine, PLO17, DPDPE and U50,488H all inhibited diarrhea in a dose-related fashion. All four compounds inhibited diarrhea at doses much below those needed to cause analgesia. Morphine s.c. and PLO17 s.c. both inhibited diarrhea at doses lower than those required to inhibit transit. DPDPE s.c. and U50,488H s.c. had no effect on transit at any dose. The antidiarrheal effects of i.c.v. morphine, i.c.v. PLO17 and i.c.v. DPDPE were antagonized by pretreatment with 1 microgram i.c.v. of naltrexone.(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1989

[3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain.

The cyclic, conformationally restricted octapeptide [3H]-[H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2] ([3H]CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of [3H]CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. [3H]CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on [3H]CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide [3H]-[D-Pen2,D-Pen5]enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific [3H]CTOP binding was inhibited by a large number of opioid or opiate ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Novel peptidic mu opioid antagonists: pharmacologic characterization in vitro and in vivo.

A series of six synthetic octapeptides, structurally related to somatostatin, demonstrate high affinity and selectivity for mu opioid receptors in radioligand binding assays. The compounds, D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), D-tetrahydroisoquinoline carboxylic acid (D-Tic)-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (D-Tic-CTP), D-Tic-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (D-Tic-CTOP) and D-Tic-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (D-Tic-CTAP), were tested in vitro and in vivo for agonist and antagonist potency and selectivity. In vitro bioassays included the guinea pig ileum, mouse vas deferens and rabbit vas deferens. In vivo tests included hotplate antinociception and gastrointestinal transit inhibition, performed in mice. In vitro, all six derivatives were competitive, highly selective mu antagonists (pA2 values from 6.4-7.9). The compounds demonstrated varying degrees of intrinsic agonist activity especially in the mouse vas deferens, the least active being CTAP and D-Tic-CTAP, which showed no mu or kappa agonist actions, and delta activity only at very high (greater than 3 microM) concentrations. In vivo, none of these compounds showed antinociceptive actions when administered i.c.v. in mice. All were competitive mu antagonists in the hotplate antinociception test.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Antagonist activity of the cyclic somatostatin analogue CTP at mu- but not delta- and kappa-opioid receptors involved in presynaptic inhibition of neurotransmitter release.

In the present study the opioid receptor antagonist properties of the conformationally constrained cyclic octapeptide D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Pen-Thr-NH2 (CTP), which is derived from somatostatin, were investigated, using in vitro functional paradigms of central mu-, delta- and kappa-opioid receptors. Activation of mu-opioid receptors by the enkephalin analogues DADLE or DAGO resulted in a strong inhibition (by 60-70%) of the (electrically evoked) release of [3H]noradrenaline (NA) from superfused cortical slices. This inhibitory effect was antagonized by CTP in a competitive fashion (pA2 value 7.7-7.9). Activation of kappa-opioid receptors by bremazocine selectively inhibited (by 45-50%) the release of [3H]dopamine (DA) from striatal slices, whereas activation of delta-opioid receptors by DADLE caused an inhibition (by 55-60%) of striatal [14C]acetylcholine (ACh) release, but neither of these inhibitory effects was affected by CTP. By itself, CTP inhibited cortical [3H]NA release (by 35-40%), but it did not affect the release of [3H]DA nor that of [14C]ACh from striatal slices. The inhibitory effect of CTP was not antagonized by naloxone. The data indicate that CTP selectively antagonizes mu-opioid receptors, involved in presynaptic inhibition of NA release in the brain. In addition, the peptide by itself causes an inhibition of NA release via a non-opioid receptor-mediated process.

Adrenergic alpha-Agonists↗
PubMed1988

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2.

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Alpha-melanocyte-stimulating hormone structure-activity studies: comparative analysis of melanotropic and CNS bioactivities.

The structure-activity relationships in vitro of alpha-MSH (alpha-melanocyte-stimulating hormone, alpha-melanotropin) analogs as determined on normal and transformed (melanoma cell) melanocyte bioassays are summarized. Specifically, the characterization of potent and metabolically stable melanotropic agonist analogs and a newly discovered antagonist of alpha-MSH are highlighted. Comparison of these data versus the known structure-activity relationships of alpha-MSH related to CNS bioactivities suggests the existence of nonclassical alpha-MSH receptor-mediated pathways or, perhaps, a yet undefined endogenous neuropeptidergic pathway(s) having different selectivities for alpha-MSH analogs. In summary, several of the alpha-MSH analogs reported here may be useful molecular probes in future strategies aimed at the identification and systematic characterization of both peripheral and central alpha-MSH receptors.

Amino Acid Sequence↗
PubMed1988

Toxicologic studies of a superpotent alpha-melanotropin, [Nle4, D-Phe7]alpha-MSH.

A toxicology study was performed in mice given a superpotent alpha melanocyte stimulating hormone (MSH) analog. This 13 amino acid derivative, [Nle4, D-Phe7]alpha-MSH or NDP-MSH, is a melanotropin which is very slowly biodegraded in vivo and is active at 1/1,000 the concentration of natural alpha-MSH. Mice were administered up to 2 mg/kg of the analog daily and weekly over 4 or 12 weeks by both topical application (in 90% DMSO) or by IP injections (in physiologic saline). At the end of this period, no toxic effects were observed in various organs, on hematologic indices, or on weight gain. A slight increase in triglyceride and platelet levels were noted in mice given the analog weekly for 12 weeks. There was no evidence of an effect on behavior nor ACTH-like endocrine actions such as elevated serum cortisol levels. Transdermal drug delivery studies performed in vitro showed reproducible diffusion of the NDP-MSH analog through full-thickness mouse skin. Approximately 0.002% to 0.05% of a 10(-4) M preparation was transdermally delivered using a DMSO/water solution or a PEG/alcohol cream base, respectively. This superpotent analog is now entering a Phase I clinical trial with possible therapeutic applications for the treatment of hypomelanotic disorders such as vitiligo and for pharmacologic tanning without the need for sunlight exposure.

Administration, Cutaneous↗
PubMed1988

Quantitative autoradiography of [3H]CTOP binding to mu opioid receptors in rat brain.

[3H]H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ([3H]CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with [3H]CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited [3H]CTOP binding with high affinity (IC50 values of 0.2-2.4 nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69, 593 were very weak inhibitors of [3H]CTOP binding (IC50 values of 234-3631 nM). Light microscopic autoradiography of [3H]CTOP binding sites revealed regions of high density (nucleus of the solitary tract, clusters in the caudate-putamen, interpeduncular nucleus, superior and inferior colliculus, subiculum, substantia nigra zona reticulata, medial geniculate, locus coeruleus and dorsal motor nucleus of the vagus) and regions of moderate labeling (areas outside of clusters in the caudate-putamen, cingulate cortex, claustrum and nucleus accumbens). The cerebral cortex (parietal) showed a low density of [3H]CTOP binding.

Animals↗
PubMed1988

Transdermal delivery of a melanotropic peptide hormone analogue.

We previously reported that topical application of [Nle4,D-Phe7]alpha-MSH, a superpotent analogue of alpha-melanocyte stimulating hormone, to mice induces a darkening of follicular melanocytes throughout the skin. We now report that the melanotropin analogue can be delivered across mouse but not rat skin in an in vitro model system. Passage of the analogue from the topically applied vehicle (polyethylene glycol) across the skin into a subcutaneous receiving vessel was demonstrated by both bioassay as well as by radioimmunoassay. The bioassay data demonstrate that percutaneous absorption of the melanotropin did not result in loss of biological activity of the peptide. The differential penetration of the peptide across rodent skin reveals that one cannot predict percutaneous absorption of a substance across the stratum corneum from studies on a single species. The present results are the first to demonstrate, by direct quantitative measurements, that a bioactive peptide can be delivered across the vertebrate integument in vitro. These studies point out the potential of a topically applied melanotropin for tanning of the skin and possibly for treatment of certain hypopigmentary disorders.

Administration, Cutaneous↗
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↗