Phosphodiesterase 4B knockout prevents skeletal muscle atrophy in rats with burn injury.

The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.

On the expansion of ribosomal proteins and RNAs in eukaryotes.

While the ribosome constitution is similar in all biota, there is a considerable increase in size of both ribosomal proteins (RPs) and RNAs in eukaryotes as compared to archaea and bacteria. This is pronounced in the large (60S) ribosomal subunit (LSU). In addition to enlargement (apparently maximized already in lower eukarya), the RP changes include increases in fraction, segregation and clustering of basic residues, and decrease in hydrophobicity. The acidic fraction is lower in eukaryote as compared to prokaryote RPs. In all eukaryote groups tested, the LSU RPs have significantly higher content of basic residues and homobasic segments than the SSU RPs. The vertebrate LSU RPs have much higher sequestration of basic residues than those of bacteria, archaea and even of the lower eukarya. The basic clusters are highly aligned in the vertebrate, but less in the lower eukarya, and only within families in archaea and bacteria. Increase in the basicity of RPs, besides helping transport to the nucleus, should promote stability of the assembled ribosome as well as the association with translocons and other intracellular matrix proteins. The size and GC nucleotide bias of the expansion segments of large LSU rRNAs also culminate in the vertebrate, and should support ribosome association with the endoplasmic reticulum and other intracellular networks. However, the expansion and nucleotide bias of eukaryote LSU rRNAs do not clearly correlate with changes in ionic parameters of LSU ribosomal proteins.

Dimers of G-protein coupled receptors as versatile storage and response units.

The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αßγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.

On the segregation of protein ionic residues by charge type.

Based on ubiquitous presence of large ionic motifs and clusters in proteins involved in gene transcription and protein synthesis, we analyzed the distribution of ionizable sidechains in a broad selection of proteins with regulatory, metabolic, structural and adhesive functions, in agonist, antagonist, toxin and antimicrobial peptides, and in self-excising inteins and intron-derived proteins and sequence constructs. All tested groups, regardless of taxa or sequence size, show considerable segregation of ionizable sidechains into same type charge (homoionic) tracts. These segments in most cases exceed half of the sequence length and comprise more than two-thirds of all ionizable sidechains. This distribution of ionic residues apparently reflects a fundamental advantage of sorted electrostatic contacts in association of sequence elements within and between polypeptides, as well as in interaction with polynucleotides. While large ionic densities are encountered in highly interactive proteins, the average ionic density in most sets does not change appreciably with size of the homoionic segments, which supports the segregation as a modular feature favoring association.

Maintenance of Y receptor dimers in epithelial cells depends on interaction with G-protein heterotrimers.

Treatment of CHO cells expressing human Y receptors (Y(1), Y(2) or Y4 subtype) with pertussis toxin results in a large decrease in functional receptors, with a preferential loss of heteropentameric assemblies of receptor dimers and G-protein trimers. This occurs in parallel to inactivation of the nucleotide site of Gi α subunits, with a half period of about 4 h. The loss could be mainly due to proteolysis at the level of recycling/perinuclear endosomes, and of receptor completion in the ER, since it is reduced by co-treatment with ammonium chloride, an inhibitor of particulate proteinases. Antagonists do not strongly decrease the heteropentameric fraction. These findings indicate that the upkeep of Y receptor dimers in epithelial cell lines depends on the association of receptor oligomers with functional Gi α subunits. This interaction could use the juxtamembrane helix 8 in the fourth intracellular domain, and could also be supported by the C-terminal helix of the third intracellular loop, as outlined in the companion review (Parker et al., Amino Acids, doi: 10.1007/s00726-010-0616-1 , 2010).

Combining neuropeptide Y and mesenchymal stem cells reverses remodeling after myocardial infarction.

Neuropeptide Y (NPY) induced reentry of differentiated rat neonatal and adult cardiomyocytes into the cell cycle. NPY also induced differentiation of bone marrow-derived mesenchymal stem cells (MSC) into cardiomyocytes following transplantation into infarcted myocardium. Rat neonatal and adult cardiomyocytes were treated in vitro with vehicle, NPY, fibroblast growth factor (FGF; 100 ng/ml), or FGF plus NPY. DNA synthesis, mitosis, and cytokinesis were determined by immunocytochemistry. NPY-induced MSC gene expression, cell migration, tube formation, and endothelial cell differentiation were analyzed. Male rat green fluorescent protein-MSC (2 x 10(6)), pretreated with either vehicle or NPY (10(-8) M) for 72 h, were injected into the border zone of the female myocardium following left anterior descending artery ligation. On day 30, heart function was assessed, and hearts were harvested for histological and immunohistochemical analyses. NPY increased 5-bromo-2'-deoxy-uridine incorporation and promoted both cytokinesis and mitosis in rat neonatal and adult myocytes. NPY also upregulated several genes required for mitosis in MSC, including aurora B kinase, FGF-2, cycline A2, eukaryotic initiation factor 4 E, and stromal cell-derived factor-1alpha. NPY directly induced neonatal and adult cardiomyocyte cell-cycle reentry and enhanced the number of differentiated cardiomyocytes from MSC in the infarcted myocardium, which corresponded to improved cardiac function, reduced fibrosis, ventricular remodeling, and increased angiomyogenesis. It is concluded that a combined treatment of NPY with MSC is a novel approach for cardiac repair.

Fragmentation and Matching of Human MicroRNA Sequences in 3'utr.

AIMS: Definition of sense and antisense microRNA matches in 3'utr. BACKGROUND: Matches of mature microRNAs (m-miRs) in human 3'utr could be traced to mutations producing fragments of original m-miR sequences without physical separation. (The m-miR matches in 5'utr and cds should be by far fewer, but could follow similar patterns). OBJECTIVE: To ascertain if the sense and antisense m-miR fragments in 3'utr occur at similar or different levels. METHODS: Frequency of sense and antisense m-miR matches in 3'utr was examined in the range of 7-22 nucleotides. RESULTS: The fragmentation occurs at gene level by mutation within one of the paired m-miRs, which upon transcription results in increased interactive capability for both former pre-micro (premir) RNA stem partners. The non-mutated stem partner can persist in 3'utr sequences, as is apparent from significant presence of miR-619-5p and miR-5096 and some conservation of 20 other simian- specific m-miR sequences. However, most of m-mir sequences in 3'utr are extensively fragmented, with low preservation of long matches. In flanks of individual m-miR embeds the mutated pre-mir positions are to a degree defined specifically. CONCLUSION: The m-mir matches of various sizes in 3'utr apparently reflect accumulation, on a phylogenetic time scale, of in-sequence point mutations. Across human 3'utr this fragmentation is significantly less for evolutionarily recent human m-miRs that originate in simians compared to human m-miRs first appearing in lower primates, and especially to human m-miRs introduced in nonprimates.

Pertussis toxin induces parallel loss of neuropeptide Y Y1 receptor dimers and Gi alpha subunit function in CHO cells.

Treatment with pertussis toxin in addition to a stable inhibition of G(i)alpha subunits of G-proteins also strongly reduced human neuropeptide Y Y(1) receptors expressed in Chinese hamster ovary (CHO) cells. This was reflected in abolition of the inhibition by Y(1) agonists of forskolin-stimulated adenylyl cyclase in intact cells, and of Y(1) agonist stimulation of GTPgammaS binding to particulates from disrupted cells. The loss of both receptor and G(i)alpha subunit function was attenuated by ammonium chloride, an inhibitor of acid proteinases, pointing to a chaperoning co-protection of active pertussis toxin-sensitive Galpha subunits and Y(1) receptors. The surface complement of the Y(1) receptor was changed a little in conditions of approximately 85% decrease of the Y(1) population, but the rate of the Y(1) receptor-linked internalization of agonist peptides was reduced about 70%. The preserved receptor fraction consisted of monomers significantly coupled to G(q)alpha subunits. The persistent pertussis toxin-insensitive internalization of agonists with the Y(1) receptor may reflect a rescue or alternative switching that could be important for cell functioning in neuropeptide Y-rich environments. The results are compatible with a loss, due to G(i)alpha subunit inactivation by the toxin, of a large Y(1) receptor reserve constituted of oligomers associating with heterotrimeric G-proteins.

Importance of a N-terminal aspartate in the internalization of the neuropeptide Y Y2 receptor.

With human neuropeptide Y Y2 receptor expressed in the Chinese hamster ovary (CHO) cells, the Asp35Ala mutation, and especially the change of Pro34Asp35 to Ala34Ala35, decrease the compartmentalization and strongly accelerate internalization of the receptor. These changes are not associated with alterations in agonist affinity, G-protein interaction, dimerization, or level of expression of the mutated receptors relative to the wildtype receptor. The proline-flanked aspartate in the N-terminal extracellular segment of the neuropeptide Y Y2 receptor thus apparently has a large role in anchoring and compartmentalization of the receptor. However, the Pro34Ala mutation does not significantly affect the embedding and cycling of the receptor.

Estrogen up-regulates neuropeptide Y Y1 receptor expression in a human breast cancer cell line.

Normal breast tissue mainly expresses the neuropeptide Y (NPY) Y2 receptor whereas primary human breast carcinomas express the Y1 receptor (Y1R) subtype. We hypothesized that activation of estrogen signaling systems plays a role in the induction of Y1R. To investigate this possibility, we used estrogen receptor-positive (ER+) human breast carcinoma cell line, MCF-7, and examined the effect of estrogen on Y1R gene expression and its signaling pathways. Saturation binding studies revealed that MCF-7 cells express high-affinity NPY receptor. NPY inhibited forskolin-stimulated adenosine 3'5'-cyclic monophosphate (cAMP) accumulation and mobilized intracellular Ca(2+) in MCF-7 cells. Chronic estrogen treatment enhanced NPY-mediated inhibition of cAMP accumulation by 4-fold and caused a significant increase in Y1R mRNA expression through ERalpha. Similarly, estrogen increased Y1R mRNA expression in T-47D (ER+) but not in MDA-MB231 or MDA-MB468 (ER-) cell lines. Cycloheximide decreased basal Y1R mRNA expression; however, it did not affect its increase by estrogen. Moreover, estrogen treatment of MCF-7 cells did not increase Y1R mRNA stability. The up-regulation of Y1R expression by estrogen is prevented by hydroxyurea but not by nocodazole or IB-MECA (cell cycle inhibitors). Lastly, NPY inhibited estrogen-induced cell proliferation through Y1R. In conclusion, MCF-7 cells express a functional Y1R coupled to both Ca(2+) and cAMP pathways. Estrogen up-regulates Y1R expression through ERalpha. This effect is independent of increased Y1R mRNA stability or new protein synthesis, and likely occurs during S phase completion of the cell cycle. Estrogen plays an important role in the up-regulation of Y1R, which in turn regulates estrogen-induced cell proliferation in breast cancer cells.

The Expansion Segments of Human 28S rRNA Match MicroRNAs Much Above 18S rRNA or Core Segments.

BACKGROUND: The size of eukaryotic 25-28S rRNAs shows a progressive phylogenetically linked increase which is pronounced in mammals, and especially in hominids. The increase is confined to specific expansion segments, inserted at points that are highly conserved from yeast to man. These segments also show a progressive increase in nucleotide bias, mostly the GC bias. Substantial parts of the large expansion segments 7, 15 and 27 of 28S rRNA are known to be exposed at the ribosome surface, with no clear association with ribosomal proteins. These segments could bind extraneous RNAs and proteins to support regulatory events. METHODS: This study examined the possible canonical matching of human 28S rRNA and 18S rRNA segments with 2586 human microRNAs. This was compared with matching of the microRNAs to sectors of 18810 human mRNAs. RESULTS: The overall matching was rather similar across 18S rRNA segments and core segments of 28S rRNA. However, the expansion segments of 28S rRNA (abbreviated ESL) collectively have a much higher (up to two-fold) capacity for the canonical association with microRNAs. This is pronounced in large ESL, and is found to strongly relate to the GC content of microRNAs. CONCLUSION: Oligonucleotides and microRNAs of high GC content through a strong canonical hydrogen bonding could have large activity in regulation of subcellular RNAs. In view of the considerable abundance of ribosomal RNAs in many mammalian tissues, ESL could constitute an important component of microRNA balance, possibly serving to lower the availability of GC-rich microRNAs (and thereby help conservation of GC-rich mRNAs).

The Expansion Segments of 28S Ribosomal RNA Extensively Match Human Messenger RNAs.

Eukaryote ribosomal RNAs (rRNAs) have expanded in the course of phylogeny by addition of nucleotides in specific insertion areas, the expansion segments. These number about 40 in the larger (25-28S) rRNA (up to 2,400 nucleotides), and about 12 in the smaller (18S) rRNA (<700 nucleotides). Expansion of the larger rRNA shows a clear phylogenetic increase, with a dramatic rise in mammals and especially in hominids. Substantial portions of expansion segments in this RNA are not bound to ribosomal proteins, and may engage extraneous interactants, including messenger RNAs (mRNAs). Studies on the ribosome-mRNA interaction have focused on proteins of the smaller ribosomal subunit, with some examination of 18S rRNA. However, the expansion segments of human 28S rRNA show much higher density and numbers of mRNA matches than those of 18S rRNA, and also a higher density and match numbers than its own core parts. We have studied that with frequent and potentially stable matches containing 7-15 nucleotides. The expansion segments of 28S rRNA average more than 50 matches per mRNA even assuming only 5% of their sequence as available for such interaction. Large expansion segments 7, 15, and 27 of 28S rRNA also have copious long (≥10-nucleotide) matches to most human mRNAs, with frequencies much higher than in other 28S rRNA parts. Expansion segments 7 and 27 and especially segment 15 of 28S rRNA show large size increase in mammals compared to other metazoans, which could reflect a gain of function related to interaction with non-ribosomal partners. The 28S rRNA expansion segment 15 shows very high increments in size, guanosine, and cytidine nucleotide content and mRNA matching in mammals, and especially in hominids. With these segments (but not with other 28S rRNA or any 18S rRNA expansion segments) the density and number of matches are much higher in 5'-terminal than in 3'-terminal untranslated mRNA regions, which may relate to mRNA mobilization via 5' termini. Matches in the expansion segments 7, 15, and 27 of human 28S rRNA appear as candidates for general interaction with mRNAs, especially those associated with intracellular matrices such as the endoplasmic reticulum.

Neuropeptide Y (NPY) Y2 receptor-selective agonist inhibits food intake and promotes fat metabolism in mice: combined anorectic effects of Y2 and Y4 receptor-selective agonists.

Peripheral administration of the endogenous Y(2) and Y(4) receptor selective agonists, PYY(3-36) and PP, have been shown to inhibit food intake and body weight gain in rodents, and to reduce appetite and caloric intake in humans. We have previously developed a long-acting, potent and highly selective Y(2) receptor selective agonist, N-alpha-Ac-[Nle(24,28), Trp(30), Nva(31), Psi(35-36)]PYY(22-36)-NH(2) (BT-48). BT-48 (ip) dose-dependently inhibited ad lib food intake and also decreased the respiratory quotient in mice during both the light and dark periods. The latter observation is indicative of enhanced fat metabolism. Moreover, BT-48 also inhibited food intake in fasted mice. Combined ip administration of BT-48 (50nmol/mouse) with a highly potent and selective Y(4) anorectic peptide, BVD-74D (50nmol/mouse), resulted in a powerful and long lasting inhibitory effect on food intake. As expected, this inhibitory effect on food intake was nearly double that exhibited by either peptide (50nmol/mouse) alone. In summary, BT-48, unlike PYY(3-36), exhibits little or no affinity to other "Y" receptors, and may therefore have a better clinical potential than PYY(3-36) for control of food intake. Moreover, it appears that treatment with a combination of Y(2) and Y(4) receptor selective agonists may constitute a more powerful approach to control food intake than treatment with either of these agonists alone.

Interaction of NPY compounds with the rat glucocorticoid-induced receptor (GIR) reveals similarity to the NPY-Y2 receptor.

The rat glucocorticoid-induced receptor (rGIR) is an orphan G protein-coupled receptor awaiting pharmacological characterization. Among known receptors, rGIR exhibits highest sequence similarity to the neuropeptide Y (NPY)-Y(2) receptor (38-40%). The pharmacological profile of rGIR was investigated using (125)I-PYY(3-36), a Y(2)-preferring radioligand and several NPY analogs. rGIR displayed a similar displacement profile as reported for the Y(2) receptor, in that the Y(2)-selective C terminus fragments of NPY and PYY (NPY(3-36) and PYY(3-36)) showed high affinity binding and activation of rGIR (low nanomolar range). The rank order potency for displacement was NPY(3-36)>PYY(3-36)=NPY>NPY(13-36)>Ac, Leu NPY(24-36)>[D-Trp(32)]-NPY>Leu(31), Pro(34)-NPY=hPP. NPY and Y(2)-selective agonists NPY(3-36) and PYY(3-36) led to significant activation of (35)S-GTPgammaS binding to rGIR transfected cells. BIIE0246, a specific Y(2) antagonist, displaced (125)I-PYY(3-36) binding to rGIR with high affinity (95nM). Activation of (35)S-GTPgammaS binding by Y(2)-selective agonist in rGIR transfected cells was also completely abolished by BIIE0246. Our data report, for the first time, an interaction of NPY ligands with rGIR expressed in vitro, and indicate similarities between GIR and the NPY-Y(2) receptor.

Canonical microRNA Matching Differs Greatly Across Groups of G-protein Coupled Receptor mRNAs.

BACKGROUND: Heptahelical G protein coupled receptors (GPCRs) support numerous sensory and metabolic functions and differ considerably in levels of expression. GPCR protein levels should link to regulation of GPCR mRNAs by microRNAs (miRs), which might significantly depend on numbers, size and GC content of the canonical antisense matches in mRNAs. These parameters of GPCR mRNAs have not been studied in detail. METHODS: Canonical matching profiles of human GPCR mRNAs and miRs were examined using segments of 7-15 nucleotides in windows shifted by one position over the entire microRNA sequence. RESULTS: Human GPCRs mRNAs within larger function-related groups have a quite homogenous matching with miRs. Both the GC content and the melting temperature (and hence also the binding energy) are appreciably higher in 5'utr compared to 3'utr matches of the same length. Increase in the GC content correlates significantly with length in the ubiquitous matches of 7-12 nucleotides. However, several GPCR groups strongly differ in overall match numbers and density. The untranslated regions of sensory receptor mRNAs, especially the olfactory and Taste-2 mRNAs, have the lowest match numbers and density and the fewest miR partners. The glucagon and frizzled families show the highest canonical matching. CONCLUSION: Partnership of GPCR mRNAs and miRs could significantly relate to the type of function of the receptor proteins, with mRNAs of the sensory receptors having the lowest and those of metabotropic GPCRs the highest targeting. This could be of interest regarding GPCR regulation by exogenous miRs.

Neuropeptide Y (NPY) Y4 receptor selective agonists based on NPY(32-36): development of an anorectic Y4 receptor selective agonist with picomolar affinity.

We have previously shown [Cys-Trp-Arg-Nva-Arg-Tyr-NH(2)](2), 1, to be a moderately selective neuropeptide Y (NPY) Y(4) receptor agonist. Toward improving the selectivity and potency for Y(4) receptors, we studied the effects of dimerizing H-Trp-Arg-Nva-Arg-Tyr-NH(2) using various diamino-dicarboxylic acids containing either di-, tri-, or tetramethylene spacers. These parallel dimers, 2A, 2B, 3, 4A, and 4B, and the corresponding linear tandem dimer and trimer analogues, 5 and 6, had enhanced selectivity and affinity for Y(4) receptors compared to 1 (Table 1). Substitution of Trp and Nva with Tyr and Leu, respectively, as in 2,7-d/l-diaminosuberic acid derivatized dimer, 7, resulted in a superior Y(4) selective agonist with picomolar affinity. Intraperitoneal (ip) injection of 7 potently inhibited food intake in fasted mice. Moreover, 7 (ip) inhibited the food intake in wild-type mice and not in Y(4)(-/-) knock-out mice, confirming that the actions of 7 on food intake are not due to global effects, but specifically mediated Y(4) receptors.

The role of polyamines in glucagon-like peptide-2 prevention of TPN-induced gut hypoplasia.

Total parenteral nutrition (TPN) of rats has been demonstrated to produce hypoplasia of gut mucosa, and to be associated with reduced immune response and elevated translocation of bacteria from gut to mesenteric lymph nodes, spleen and liver. Treatment of rats being maintained on TPN with the proglucagon fragment, glucagon-like peptide-2 (GLP-2), has been shown to totally prevent small intestine mucosal hypoplasia. In the present study, we found that depletion of polyamines with alpha-difluromethylornithine (DFMO) significantly reduced the efficacy of GLP-2 in preserving gut mucosa in rats maintained on TPN for 8 days. Co-infusion of GLP-2 with TPN prevented loss of protein and mucosa in duodenum, jejunum and ileum, but not in colon. Addition of DFMO to the infusate prevented the protective effects of GLP-2 in the duodenum and jejunum. In the jejunum, putrescine and spermidine were reduced in DFMO-treated rats, while the ileum exhibited reductions of these polyamines in rats infused with TPN or TPN plus GLP-2. DFMO infusion further reduced these polyamines in the ileum, while levels of spermine were increased. Concentrations of ornithine decarboxylase were elevated in jejunum of rats infused with TPN or TPN plus GLP-2, but were reduced significantly in DFMO-treated rats. These results suggest that normal levels of polyamines are necessary for the expression of GLP-2-induced hyperplasia. Differential effects of GLP-2 and DFMO across gut segments may relate to regional differences in proliferative and anti-apoptotic effects of the treatments.

Ghrelin stimulates food intake and growth hormone release in rats with thermal injury: synthesis of ghrelin.

Ghrelin, a 28-residue octanoylated peptide recently isolated from the stomach, exhibits anti-cachectic properties through regulating food intake, energy expenditure, adiposity, growth hormone secretion and immune response. Burn injury induces persistent hypermetabolism and muscle wasting. We therefore hypothesized that ghrelin may also play a role in the pathophysiology of burn-induced cachexia. Overall ghrelin expression in the stomach over 10 days after burn was significantly decreased (p = 0.0003). Total plasma ghrelin was reduced 1 day after burn. Thus, changes in ghrelin synthesis and release may contribute to burn-induced dysfunctions. Ghrelin (30 nmol/rat, i.p.) greatly stimulated 2 h food intake in rats on five separate days after burn and in control rats. On post-burn day 15, plasma growth hormone levels were significantly lower than in controls, and this was restored to normal levels by ghrelin (10 nmol/rat, i.p.). These observations suggest that ghrelin retains its ability to favorably modulate both the peripheral anabolic and the central orexigenic signals, even after thermal injury despite ongoing changes due to prolonged and profound hypermetabolism, suggesting that long-term treatment with ghrelin may attenuate burn-induced dysfunctions.

Neuropeptide Y as a partial agonist of the Y1 receptor.

In absence of receptor cycling, human/rat neuropeptide Y was found to persistently occupy the guinea pig neuropeptide Y Y1 receptors expressed on the surface of Chinese hamster ovary (CHO) cells (IC50 approximately 8 nM); a lasting occupancy was also evident with active receptor cycling. A similar blockade was obtained with the human neuropeptide Y Y1 receptor (in CHO or SK-N-MC cells). Peptidic antagonists GR238118 (1229U91) and VD-11 blocked the Y1 receptor in the same molarity range. A neuropeptide Y-related Y1 agonist, (Leu31Pro34) human neuropeptide Y, also strongly adhered to the Y1 site. Similar blockade-like occupancy by neuropeptide Y was found with particulates from Y1-expressing CHO cells, and with native neuropeptide Y Y1 receptors of rat synaptosomes. Peptide YY and a related Y1-selective agonist, (Leu31Pro34) human peptide YY, showed a much less stable binding to the neuropeptide Y Y1 receptor with either the intact cells or particulates. The Y1 binding of neuropeptide Y was also less sensitive to chaotropic agents and guanine nucleotides than the binding of peptide YY, indicating a larger stability for association of neuropeptide Y with the receptor. Inhibition of forskolin-stimulated adenylyl cyclase showed a distinctly attenuating agonism for neuropeptide Y, with an activity similar to peptide YY below 1 nM, but considerably lower above 3 nM of the peptides. This activity was largely exerted via pertussis toxin-sensitive G-proteins of Y1-CHO cells. Our findings indicate that signaling by neuropeptide Y via its Y1 receptor could be self-restricting at higher levels of the peptide, in relation to a strong association of the agonist with the Y1 binding site.

Lithium inhibits internalization and endosomal processing of both neuropeptide Y (NPY) Y1 and transferrin receptors.

Low concentrations of Li+ reduce the rate of internalization of neuropeptide Y (NPY) Y1 receptors [M.S. Parker, S.L. Parker, J.K. Kane, Internalization of neuropeptide Y Y1 and Y5 and of pancreatic polypeptide Y4 receptors is inhibited by lithium in preference to sodium and potassium ions, Regul. Pept., 118 (2004) 67-74]. This Li+-induced decrease in Y1 receptor internalization could be alleviated by Y1 receptor agonists. As shown by fractionation on Percoll gradients, lithium treatment induces a concentration-related decrease of intermediate and higher endosomal densities that contain the internalized Y1 ligand-receptor complex. This indicates an inhibition of endosome processing and maturation. Internalization of human transferrin shows [Li+] sensitivity similar to that of the Y1 receptor, and a similar Li+-induced decrease in endosomal processing. Lithium treatment thus decreases activity of the endosome system shared in the recycling endocytosis of the Y1 and transferrin receptors.