Follicle growth and oocyte developmental competence in cows with liver damage.

Follicle growth, oocyte quality or oocyte growing environment (follicular fluid) were evaluated in cows with severe liver damage (haemorrhage, telangiectasis, cholangitis and abscess) that were visually diagnosed at the slaughterhouse. Holstein cows aged 40-90 months with either a healthy liver (HL cow) or damaged liver (DL cow) were selected as donors. Follicle development kinetics was evaluated by counting the follicles at various developmental stages. In addition, the biochemical characteristics of the follicular fluids, developmental competence of preantral follicles cultured for 16 days in vitro and the ability of oocytes to develop to the blastocyst stage 8 days after fertilization were examined. DL cows had fewer secondary follicles than HL cows, and the correlation between the number of secondary follicles and the number of primary follicles differed among DL and HL cows. The follicular fluid of DL cows contained significantly lower levels of albumin and a higher total protein content than that of HL cows. Oocyte nuclear maturation assessed at 5, 16 and 21 h after beginning of culture was slower in DL cows than in HL cows, although the final maturation rates did not differ. The rate of polyspermic fertilization was significantly higher and the proportion of cleavage at 48 h after insemination and blastulation lower in DL cows compared with HL cows. When preantral follicles were cultured in vitro, the rate of follicles with normal morphology was lower in DL cows than in HL cows. These findings suggest that the kinetics of folliculogenesis differ among DL and HL cows and the developmental ability of preantral follicles and oocytes is lower in DL cows than in HL cows.

Novel antagonists for proteinase-activated receptor 2: inhibition of cellular and vascular responses in vitro and in vivo.

BACKGROUND AND PURPOSE: Proteinase-activated receptor 2 (PAR(2)) is a G-protein coupled receptor associated with many pathophysiological functions. To date, the development of PAR(2) antagonists has been limited. Here, we identify a number of novel peptide-mimetic PAR(2) antagonists and demonstrate inhibitory effects on PAR(2)-mediated intracellular signalling pathways and vascular responses. EXPERIMENTAL APPROACH: The peptide-mimetic compound library based on the structures of PAR(2) agonist peptides were screened for inhibition of PAR(2)-induced calcium mobilisation in human keratinocytes. Representative compounds were further evaluated by radioligand binding and inhibition of NFkappaB transcriptional activity and IL-8 production. The vascular effects of the antagonists were assessed using in vitro and in vivo models. KEY RESULTS: Two compounds, K-12940 and K-14585, significantly reduced SLIGKV-induced Ca(2+) mobilisation in primary human keratinocytes. Both K-12940 and K-14585 exhibited competitive inhibition for the binding of a high-affinity radiolabelled PAR(2)-ligand, [(3)H]-2-furoyl-LIGRL-NH(2), to human PAR(2) with K(i) values of 1.94 and 0.627 microM respectively. NFkappaB reporter activity and IL-8 production were also significantly reduced. Furthermore, relaxation of rat-isolated aorta induced by SLIGRL-NH(2) was inhibited competitively by K-14585. K-14585 also significantly lowered plasma extravasation in the dorsal skin of guinea pigs and reduced salivation in mice. CONCLUSIONS AND IMPLICATIONS: K-12940 and K-14585 antagonized PAR(2) competitively, resulting in inhibition of PAR(2)-mediated signalling and physiological responses both in vitro and in vivo. These peptide-mimetic PAR(2) antagonists could be useful in evaluating PAR(2)-mediated biological events and might lead to a new generation of therapeutically useful antagonists.

Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase beta in human endothelial cells.

BACKGROUND AND PURPOSE: Up-regulation of proteinase-activated receptor-2 (PAR2) is a factor in a number of disease states and we have therefore examined the signalling pathways involved in the expression of the receptor. EXPERIMENTAL APPROACH: We investigated the effects of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), trypsin and the PAR2 activating peptide, 2-furoyl(2f)-LIGKV-OH on both mRNA and functional expression of PAR2 in human umbilical vein endothelial cells (HUVECs). The effect of specific chemical inhibitors and dominant negative adenovirus constructs of the mitogen-activated protein kinase (MAPK) cascade and the nuclear factor kappa B (NF-kappaB) signalling pathway was assessed. Methods included semi-quantitative and quantitative RT-PCR, [(3)H]inositol phosphate (IP) accumulation and Ca(2+)-dependent fluorescence. KEY RESULTS: The above agonists induced both mRNA and functional expression of PAR2; PAR4 mRNA, but not that for PAR1 or PAR-3, also increased following TNFalpha treatment. Inhibition of p38 MAP kinase reduced PAR2 and PAR4 expression, whilst inhibition of MEK1/ERK/JNK was without effect. A similar dependency upon p38 MAP kinase was observed for the expression of PAR4. TNFalpha -induced enhancement of PAR2 stimulated [(3)H]-inositol phosphate accumulation (IP) and Ca(2+) signalling was abolished following SB203580 pre-treatment. Infection with adenovirus encoding dominant-negative IKKbeta (Ad.IKKbeta(+/-)) and to a lesser extent dominant-negative IKKalpha (Ad.IKKalpha(+/-)), substantially reduced both control and IL-1beta- induced expression of both PAR2 and PAR4 mRNA and enhancement of PAR2-stimulated IP accumulation and Ca(2+) mobilisation. CONCLUSIONS AND IMPLICATIONS: These data reveal for the first time the signalling events involved in the upregulation of both PAR2 and PAR4 during pro-inflammatory challenge.

Effects of adenosine on adhesion molecule expression and cytokine production in human PBMC depend on the receptor subtype activated.

BACKGROUND AND PURPOSE: Adenosine suppresses immune responses through adenosine(2A) (A(2A)) receptors, by raising intracellular cAMP. Interleukin (IL)-18 up-regulates the expression of intercellular adhesion molecule (ICAM)-1 on monocytes, leading to production of pro-inflammatory cytokines such as IL-12, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by human peripheral blood mononuclear cells (PBMC). We have previously demonstrated that elevation of cAMP inhibits this IL-18-induced expression of adhesion molecules. In the present study, we examined the effect of adenosine on the IL-18-induced up-regulation of ICAM-1 on human monocytes and production of IL-12, IFN-gamma and TNF-alpha by PBMC. EXPERIMENTAL APPROACH: The expression of ICAM-1 was examined by flow cytometry. IL-12, IFN-gamma and TNF-alpha were determined by ELISA assay. KEY RESULTS: Adenosine inhibited the IL-18-induced up-regulation of ICAM-1 on human monocytes and it abolished the IL-18-enhanced production of IL-12, IFN-gamma and TNF-alpha. While an A(2A) receptor antagonist reversed the action of adenosine, an A(1) or A(3) receptor antagonist enhanced them. An A(2A) receptor agonist, CGS21680, mimicked the effects of adenosine and its effects were abolished not only by the A(2A) receptor antagonist but also by A(1) or A(3) receptor agonists. Activation via A(2A) receptors resulted in elevation of cAMP in monocytes, whereas the stimulation of A(1) or A(3) receptors inhibited it, suggesting that intracellular signal transduction following ligation of A(2A) receptors might be blocked by activation of A(1) or A(3) receptors. CONCLUSIONS AND IMPLICATIONS: Adenosine differentially regulates IL-18-induced adhesion molecule expression and cytokine production through several subtypes of its receptors.

Proteinase-activated receptor-2-mediated activation of stress-activated protein kinases and inhibitory kappa B kinases in NCTC 2544 keratinocytes.

In this study we examined the regulation of the stress-activated protein (SAP) kinases and inhibitory kappa B kinases (IKKs) through stimulation of the novel G-protein-coupled receptor proteinase-activated receptor-2 in the human keratinocyte cell line NCTC2544. Trypsin and the peptide SLIGKV stimulated a time-dependent increase in both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activity. Trypsin also stimulated NF kappa B-DNA binding and the activation of the upstream kinases IKK alpha and -beta. Phorbol 12-myristate 13-acetate also strongly activated both SAP kinases and IKK isoforms, suggesting the potential for a protein kinase C-mediated regulatory mechanism underlying the effects of trypsin. Pre-incubation with selective protein kinase C (PKC) inhibitors GF109203X and Gö6983, or transfection of dominant negative (DN)-PKC alpha, abolished phorbol 12-myristate 13-acetate-mediated c-Jun N-terminal kinase activity, although it only partially inhibited the response to trypsin. In contrast, Gö6983 reduced trypsin-stimulated p38 mitogen-activated protein kinase activity to a greater extent than GF109203X, although DN-PKC alpha or PKC zeta had no substantial effect. Additionally, inhibitors of PKC partially reduced trypsin-stimulated IKK alpha activity but abolished that of IKK beta, whereas DN-PKC alpha but not DN-PKC zeta substantially reduced trypsin-stimulated Flag-IKK beta activity. This study shows for the first time proteinase-activated receptor-2-mediated stimulation of both SAP kinase and IKK signaling and differing roles for PKC isoforms in the regulation of each pathway.

VUF-K-8788, a periphery-selective histamine H1 antagonist with anti-pruritic activities.

The pharmacological properties of 7-[3-[4-(2-quinolinylmethyl)-1-piperazinyl]-propoxy]-2,3-dihydro-4H-1,4-benzothiazin-3-one (VUF-K-8788) were investigated in vitro and in vivo. VUF-K-8788 inhibited [3H]-mepyramine from binding to the cell membrane of lung parenchyma (Ki value: 5.0 nM) and the histamine-induced contraction of isolated guinea pig ileum (pA2: 9.71) without affecting ileal contractions induced by acetylcholine, serotonin, KCl and BaCl2. The increase of vascular permeabilities induced by histamine and passive cutaneous anaphylaxis (PCA) in guinea pigs were inhibited by VUF-K-8788 in a dose-dependent fashion (ED50: 0.24 and 0.26 mg/kg, p.o., respectively). Moreover, the anti-histaminic effect of VUF-K-8788 was also observed in rats. In experiments on the effects on the central nervous system, VUF-K-8788 at 1 mg/kg, p.o. hardly antagonized the H1 receptor at all in the cerebral cortex of guinea pigs. VUF-K-8788 inhibited the PCA-induced scratching behavior completely without affecting thiopental-induced sleep in mice. These results suggested that VUF-K-8788 would be useful in the treatment of allergic disorders such as atopic dermatitis and eczema.

Proteinase-activated receptors.

Proteinase-activated receptors are a recently described, novel family of seven-transmembrane G-protein-coupled receptors. Rather then being stimulated through ligand receptor occupancy, activation is initiated by cleavage of the N terminus of the receptor by a serine protease resulting in the generation of a new tethered ligand that interacts with the receptor within extracellular loop-2. To date, four proteinase-activated receptors (PARs) have been identified, with distinct N-terminal cleavage sites and tethered ligand pharmacology. In addition to the progress in the generation of PAR-1 antagonists, we describe the role of thrombin in such processes as wound healing and the evidence implicating PAR-1 in vascular disorders and cancer. We also identify advances in the understanding of PAR-1-mediated intracellular signaling and receptor desensitization. The cellular functions, signaling events, and desensitization processes involved in PAR-2 activation are also assessed. However, other major aspects of PAR-2 are highlighted, in particular the ability of several serine protease enzymes, in addition to trypsin, to function as activators of PAR-2. The likely physiological and pathophysiological roles for PAR-2 in skin, intestine, blood vessels, and the peripheral nervous system are considered in the context of PAR-2 activation by multiple serine proteases. The recent discovery of PAR-3 and PAR-4 as additional thrombin-sensitive PARs further highlights the complexity in assessing the effects of thrombin in several different systems, an issue that remains to be fully addressed. These discoveries have also highlighted possible PAR-PAR interactions at both functional and molecular levels. The future identification of other PARs and their modes of activation are an important future direction for this expanding field of study.