AIM:The quantification of newly formed bone in experimental defect models is a problem in various experimental set-ups. Several methods have been described to evaluate and quantify the regeneration of newly formed bone in various animal models. Most methods only describe the amount of regenerated tissue on a semi-quantitative level, the results significantly depend on the subjective rating of the observer and such evaluation methods have not been validated in terms of objectivity and reliability. The aim of the present study was to introduce a novel evaluation method for the accurate quantification of bone regeneration on digital X-ray images using a freely available digital image software analysis programme (GIMP, GNU General Public Licence).METHODS:The method introduced here contains 5 steps: standardisation of size and colour, determination of range of interest (ROI), defining different qualities of mineralisation, pixel analysis with histogram function, similar to the Hondsfield index, and quantification. In order to evaluate the objectivity and reliability, the quantification method was compared to semi-quantitative scores described by Mosheiff and Werntz for inter- and intraobserver variability. Six observers were asked to determine bone regeneration in 16 X-ray images of 2 different animal models. In order to describe intraobserver variability, the evaluation was repeated after a period of 4 weeks. Statistical analysis including determination of intra- and interobserver variability (Bland-Altman coefficient of reproduction) was performed using SAS software.RESULTS:For both experimental set-ups analysed in this project (rabbit and sheep bone defects), the objectivity was significantly higher in the GIMP-based evaluation compared to the evaluation according to Mosheiff and Werntz using the Bland-Altman coefficient (rabbit: GIMP: 0.095, Mosheiff: 0.272, Werntz: 0.283; sheep: GIMP: 0.098, Mosheiff: 0.658, Werntz: 0.668). Analogous results were obtained for reliability (rabbit: GIMP: 0.086, Mosheiff: 0.221, Werntz: 0.385; sheep: GIMP: 0.102, Mosheiff: 0.339, Werntz: 0.623).CONCLUSION:This quantification method introduced here has proved to be a reliable and "easy-to-use" tool in order to perform objective quantification of bone regeneration in 2 different experimental set-ups. It offers a more detailed and quantitative way for precise determination of regenerated tissue and is characterised by higher objectivity and reliability compared to other semi-quantitative evaluation methods. The objectivity seems to be independent of the animal model to which the method is applied.

Background and purpose: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF1) ligands.Experimental approach: We systematically substituted urocortin, a natural peptide agonist of CRF1, with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF1 to Gs-and G(i)-protein in human embryonic kidney cells, using receptor binding, [S-35]-GTPgS binding stimulation, and cAMP accumulation assays.Key results: Native ligands stimulated G(s) and G(i) activation through CRF1, resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6 -15 led, dependent on the position and nature of the substituent, to ligands that conserved G(s) activity, but were devoid of G(i) activity, only stimulating cAMP accumulation, and competitively antagonized the G(i) activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated G(s) and G(i), as urocortin did.Conclusions and implications: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF1. This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.

large extracellular N-terminal domains (NTs) of class B G protein-coupled receptors serve as major ligand binding sites. However, little is known about the ligand requirements for interactions with these receptor domains. Recently, we have shown that the most potent CRF receptor agonist urocortin 1 (Ucn1) has two segregated receptor binding sites Ucn1(1-21) and Ucn1(32-40). For locating the receptor domains interacting with these two sites, we have investigated the binding of appropriate Ucn1 analogues to the receptor N-termini compared to the corresponding full-length receptors. For this purpose receptor NTs of CRF(rat) subtypes 1 and 2(a) without their signal sequences were overexpressed in Escherichia coli and folded in vitro. For CRF2(a)-rNT, which bears five cysteine residues (C2-C6), the disulfide arrangement C2-C5 and C4-C6 was found, leaving C3 free. This is consistent with the disulfide pattern of CRF1-rNT, which has six cysteines and in which C1 is paired with C3. Binding studies of N-terminally truncated or C-terminally modified Ucn1 analogues demonstrate that it is the C-terminal part, Ucn1(11-40), that binds to receptor NT, indicating a two-domain binding mechanism for Ucn binding to receptor NT. Since the binding of Ucn1 to the juxtamembrane domain has been shown to be segregated from binding to the receptor N-terminus [Hoare et al. (2004) Biochemistry 43, 3996-4011], a third binding domain should exist, probably comprising residues 8-10 of Ucn, which particularly contribute to a high-affinity binding to full-length receptors but not to receptor NT.

The regulation of G protein activation by the rat corticotropin-releasing factor receptor type 1 (rCRFR1) in human embryonic kidney (HEK) 293 (HEK-rCRFR1) cell membranes was studied. Corresponding to a high and low affinity ligand binding site, sauvagine and other peptidic CRFR1 ligands evoked high and low potency responses of G protein activation, differing by 64-fold in their EC50 values as measured by stimulation of [S-35]GTPgammaS binding. Contrary to the low potency response, the high potency response was of lower GTPgammaS affinity, pertussis toxin (PTX)-insensitive, and homologously desensitized. Distinct desensitization was also observed in the adenylate cyclase activity, when its high potency stimulation was abolished and the activity became low potently inhibited by sauvagine. From these results and immunoprecipitation of [S-35]GTPgammaS-bound Galpha(s) and Galpha(i) subunits it is concluded that the high and low potency [S-35]GTPgammaS binding stimulation reflected coupling to G(s) and G(i) proteins, respectively, only G(s) coupling being homologously desensitized. Immunoprecipitation of [S-35]GTPgammaS-bound Galpha(q/11) revealed additional coupling to G(q/11), which also was homologously desensitized. Although Galpha(q/11) coupling was PTX-insensitive, half of the sauvagine-stimulated accumulation of inositol phosphates in the cells was PTX-sensitive, suggesting involvement of G(i) in addition to G(q/11) in the stimulation of inositol metabolism. It is concluded that CRFR1 signals through at least two different ways, one leading to G(s)- and G(q/11)-mediated signaling steps and desensitization and another leading to G(i)-mediated signals without being desensitized. Furthermore, the concentrations of the stimulating ligand and GTP and desensitization may be part of a regulatory mechanism determining the actual ratio of the coupling of CRFR1 to different G proteins.

Using confocal laser scanning microscopy we investigated the Ca(2+) distribution in single corticotropin releasing factor- and urocortin-stimulated human skin cells. The models tested included melanoma cells, neonatal melanocytes and keratinocytes, and immortalized HaCaT keratinocytes. The changes in intracellular Ca(2+) signal intensities observed after stimulation of different cell types with corticotropin releasing factor and urocortin showed that: (1) the increase of intracellular Ca(2+) concentration was caused by a Ca(2+) influx (inhibition by EGTA); (2) this Ca(2+) influx took place through voltage-activated Ca(2+) ion channels (inhibition by d-cis-diltiazem, verapamil) and (3) cyclic nucleotide-gated ion channels were not involved in this process (no effect of Mg(2+)). The effects were also observed at very low peptide concentrations (10(-13) M) with no apparent linear correlation between peptide dosage and increase of fluorescence intensity, which implied co-expression of different corticotropin releasing factor receptor forms in the same cell. Immortalized (HaCaT) keratinocytes exhibited the strongest differential increases of a Ca(2+) fluorescence after peptide-stimulation. Corticotropin releasing factor induced Ca(2+) flux into the cytoplasm, while urocortin Ca(2+) flux into the nucleus with a remarkable oscillatory effect. The latter indicated the presence of an intracellular urocortin-induced signal transduction pathway that is unique to keratinocytes.

The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction, Since N- and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N- and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible E-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC50: 100-300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC50:0.6-50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.

ABSTRACT: The classical neuroendocrine pathway for response to systemic stress is by hypothalamic release of corticotropin releasing hormone (CRH), subsequent activation of pituitary CRH receptors (CRH‐R), and production and release of proopiomelanocortin (POMC) derived peptides. It has been proposed that an equivalent to the hypothalamic‐pituitary‐adrenal axis functions in mammalian skin, in response to local stress (see Reference 1 ). To further define such system we used immunocytochemistry, RP‐HPLC separation, and RIA techniques, in rodent and human skin, and in cultured normal and malignant melanocytes and keratinocytes. Production of mRNA for CRH‐R1 was documented in mouse and human skin using RT‐PCR and Northern blot techniques; CRH binding sites and CRH‐R1 protein were also identified. Addition of CRH to immortalized human keratinocytes, and to rodent and human melanoma cells induced rapid, specific, and dose‐dependent increases in intracellular Ca 2+ . The latter were inhibited by the CRH antagonist α‐helical‐CRH(9–41) and by the depletion of extracellular calcium with EGTA. CRH production was enhanced by ultraviolet light radiation and forskolin (a stimulator for intracellular cAMP production), and inhibited by dexamethasone. Thus, evidence that skin cells, both produce CRH and express functional CRH‐R1, supports the existence of a local CRH/CRH‐R neuroendocrine pathway that may be activated within the context of a skin stress response system .

We demonstrate the presence and hair cycle-dependent expression of corticotropin-releasing factor (CRF) and CRF receptors (CRF-R) in C57BL/6 mouse skin. To correlate this with a physiological, developmentally controlled tissue remodeling process, we have analyzed CRF and CRF-R expression during defined stages of the murine hair cycle with its rhythmic changes between growth (anagen), regression (catagen), and resting (telogen). Using reversed-phase HPLC combined with two independent anti-CRF radioimmunoassays, we have identified CRF in murine skin. Maximal CRF levels were found in anagen III-IV skin, and minimal values were detected in catagen and telogen skin. By immunofluorescence, maximal CRF immunoreactivity (CRF-IR) was seen in the basal epidermis, nerve bundles of skin, the outer root sheath and matrix region of anagen IV-VI follicles, and in defined sections of their perifollicular neural network, whereas catagen and telogen skin displayed minimal CRF-IR. Using quantitative autoradiography and 125I-CRF as a tracer, high-affinity binding sites for CRF were detected in murine skin. The highest density of specific binding sites was detected in the panniculus carnosus, the epidermis, and the hair follicle. CRF-R type 1 (CRF-R1) IR was detected by immunohistology mainly in the outer root sheath, hair matrix, and dermal papilla of anagen VI follicles, as well as in the inner and outer root sheaths of early catagen follicles. CRF-R1 expression was also hair cycle dependent. Therefore, in normal murine skin, the CRF-CRF-R signaling system may operate as an additional neuroendocrine pathway regulating skin functions, possibly in the context of cutaneous stress responses.

The highly potent and efficacious μ-opioid agonist fentanyl was SC infused into rats with submaximal analgesic doses (0–1.14 μmol/kg/day) continuously for 8 days, checked by the constant daily urinary recovery of intact drug (0.43 ± 0.031% of the daily dose). Tail-flick latencies measured at 24 (day 1) and 48 h (day 2) after starting the infusion were increased in a dose-dependent fashion compared with those before the infusion (day 0). However, at day 8, the latencies were increased only weakly, not significantly, revealing tolerance to the antinociceptive activity of fentanyl. Fentanyl at all doses showed no significant effect on the capacity (Bmax) and affinity (Kd) of the μ-opioid receptor binding of DAMGO to whole brain (Bmax 126.2 ± 3.00 fmol/mg protein, Kd 1.00 ± 0.04 nM) and spinal cord (Bmax 48.24 ± 2.71 fmol/mg protein, Kd 1.93 ± 0.13 nM) membranes gained from the rats after killing them at day 8. Gpp(NH)p increased the Kd for brain and spinal cord sites by 3.09 and 2.65, respectively, independent of the fentanyl dose. The infusion with fentanyl did not alter the basal and forskolin-stimulated adenylate cyclase activity in the whole brain membranes, nor did it change the inhibition of the forskolin-stimulated activity by DAMGO. It is concluded that, in rats, constant long-term body levels of highly potent μ-agonists result in a tolerant state that, however, does not produce overall changes in the parameters of their specific receptor sites in the CNS, i.e., receptor capacity and affinity, and in the events closely related to them, i.e., their regulation by GTP and of adenylate cyclase. This does not exclude such possible changes to be restricted to specific regions in the CNS.

In order to determine which amino acid side chains of ovine corticotropin releasing factor (oCRF) are most sensitive to alterations with respect to receptor binding and activation, we synthesized a single-point replacement set by replacing each residue by a similar, preferably proteinogenic amino acid, maintaining a minimal change of character at each position (Ser by Thr, Gln by Asn, Glu by Asp, Arg by Lys, and vice versa, Pro by N-MeAla, Ile by Leu, Leu by Nle, Phe by Trp, His by Ala, Val by Leu, Met by Nle, Ala by Leu). In general, any loss in the biological potency by a single-point substitution in oCRF parallels a decrease in receptor binding, indicating that, in contrast to previous suggestions, there is no specific side chain in the peptide that is more responsible for receptor activation than for receptor binding. In addition to Arg(16), Ala(31), and Arg(35), amino acid residues in the N-terminal sequence (5-14) were found to be sensitive to alteration, demonstrating their particular importance for the receptor interaction of CRF agonists. Most of the analogs tested exhibited agonistic potencies in an in vitro pituitary cell culture assay at a concentration of 0.3 nM, and all analogs showed full agonistic potency at 1 microM. In contrast to the results of an alanine replacement study, the strongest decrease in receptor binding and biological potency was observed for analogs with substitutions of hydrophilic amino acids Ser(7), Arg(16), Glu(17), or Asn(34). In the case of Ser(7) and Arg(16), side chain specific interactions with the receptor may be required for high affinity. Alanine replacements at positions 17 or 34 resulted in analogs that were as potent as oCRF, while replacement of Glu(17) by Asp or Asn(34) by Gln caused a dramatic loss in potency, thereby suggesting an important effect at sterically or conformationally sensitive positions. In contrast to corresponding alanine analogs which exhibited a significant loss in biological potency, slight alterations of lipophilic side chains at positions 6, 12, or 38 did not cause a significant reduction of receptor binding and activation, indicating that it is not specific side chains but rather lipophilicity which is essential at these positions. Indeed, replacement of Phe(12) by Trp provides an agonist with significantly increased receptor binding and biological potency.

The potencies of the corticotropin-releasing hormone (CRH) agonistic peptides oCRH, h/rCRH, frog sauvagine, and carp urotensin I and of the antagonistic peptide alpha-helical CRH9-41 were compared in 3 different in vitro assays: (a) receptor binding to rat brain membranes; (b) release of ACTH/beta-endorphin from rat pituitary cells; and (c) relaxation of rat mesenteric small arteries. From their potency profiles, especially from the high potency of sauvagine relative to CRH in the relaxation assay, it is concluded that the receptors mediating the hypotensive action of systemic CRH in Vascular smooth muscle are different from those in the pituitary and brain, and may be identical or very similar to the recently cloned new CRH receptor type 2.

As β-turn-inducing motif, 4-(N-Fmoc-2-aminoethyl)-6-dibenzofuranpropionic acid was synthesized and incorporated into the corticotropin-releasing factor. The effect on structural and biological properties was investigated.

BACKGROUND In vitro, some neuropeptides, including the tachykinin, substance P (SP), act as growth factors. The cyclic growth of the richly innervated hair follicle offers a model for probing such functions in a complex, developmentally regulated tissue interaction system under physiologic conditions. Dissecting the role of neuropeptides in this system may also reveal as yet obscure neural mechanisms of hair growth control. EXPERIMENTAL DESIGN The neuropeptide-releasing neurotoxin, capsaicin was injected intradermally, or SP slow-release formulations were implanted subcutaneously in the back skin of C57BL/6 mice with all follicles in the resting stage of the hair cycle (telogen) in order to see whether this induced hair growth (anagen). In addition, the endogenous SP skin concentration and the activity of the main SP-degrading enzyme, neutral endopeptidase, were determined during the induced murine hair cycle by high performance liquid chromatography-controlled radioimmuno-assay (SP) or by fluorometry (neutral endopeptidase). RESULTS Both capsaicin and SP induced significant hair growth (anagen) in the back skin of telogen mice. This was associated with substantial mast cell degranulation. The endogenous SP skin concentration showed significant, hair cycle-dependent fluctuations during the induced murine hair cycle, which were largely independent of the activity of neutral endopeptidase. CONCLUSIONS SP may play a role in the neural control of hair growth. Whereas this pilot study does not address the underlying mechanisms of action, it demonstrates that SP has potential as a hair growth-stimulatory agent in vivo, and serves as a basis for exploring the role of tachykinins in epithelial-mesenchymal-neuroectodermal interaction systems like the hair follicle.

This report investigates the effect of various levels of medium-chain/fish oil structured triglycerides on protein and energy metabolism in hypermetabolic rats. Male Sprague-Dawley rats (192 to 226 g) were continuously infused with isovolemic diets that provided 200 kcal/kg per day and 2 g of amino acid nitrogen per kilogram per day. The percentage of nonnitrogen calories as structured triglyceride was varied: no fat, 5%, 15%, or 30%. A 30% long-chain triglyceride diet was also provided as a control to compare the protein-sparing abilities of these two types of fat. Nitrogen excretion, plasma albumin, plasma triglycerides, and whole-body and liver and muscle protein kinetics were determined after 3 days of feeding. Whole-body protein breakdown, flux, and oxidation were similar in all groups. The 15% structured triglyceride diet maximized whole-body protein synthesis (p < .05). Liver fractional synthetic rate was significantly greater in animals receiving 5% of nonprotein calories as structured triglyceride (p < .05). Muscle fractional synthetic rate was unchanged. Plasma triglycerides were markedly elevated in the 30% structured triglyceride-fed rats. The 30% structured triglyceride diet maintained plasma albumin levels better than those diets containing no fat, 5% medium-chain triglyceride/fish oil structured triglyceride, or 30% long-chain triglycerides. Nitrogen excretion was lower in animals receiving 30% of nonnitrogen calories as a structured triglyceride than in those receiving 30% as long-chain triglycerides, but this difference did not reach statistical significance (p = .1). These data suggest that protein metabolism is optimized when structured triglyceride is provided at relatively low dietary fat intakes.