25 research outputs found

    Pituitary adenylate cyclase activating polypeptide concentrations in the sheep mammary gland, milk, and in the lamb blood plasma after suckling

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    Pituitary adenylate cyclase activating polypeptide (PACAP) is involved in development and reproduction. We previously described elevated PACAP levels in the milk compared to the plasma, and the presence of its specific PAC1 receptor in the mammary gland. This study aimed to determine PACAP and vasoactive intestinal peptide (VIP) levels in female suckling lambs compared to ewe plasma and mammary gland, as well as their age-dependent alterations. mRNA expressions of PACAP, VIP, PAC1 receptor and brain-derived neurotrophic factor (BDNF) were quantified in the milk whey and mammary gland. PACAP38-like immunoreactivity (PACAP38-LI) was measured in plasma, milk whey and mammary gland by radioimmunoassay, VIP-LI by enzyme-linked immunoassay. PACAP38-LI was 5, 6 times higher in the milk compared to the plasma of lactating sheep. It significantly increased in the lamb plasma 1 h, but returned to basal level 2 h after suckling. However, VIP mRNA was not present in the mammary gland, we detected the VIP protein in the milk whey. BDNF mRNA significantly decreased with age to approximately 60% and 25% in the 3- and 10-year-old sheep respectively, compared to the 3-month-old lambs. No differences were found between mammary and jugular vein plasma PACAP and VIP concentrations, or during the daily cycle. We propose a rapid absorption of PACAP38 from the milk and/or its release in suckling lambs. PACAP accumulated in the milk might be synthesized in the mammary gland or secreted from the plasma of the mothers. PACAP is suggested to have differentiation/proliferation promoting and immunomodulatory effects in the newborns and/or a local function in the mammary gland

    Hemokinin-1 as a Mediator of Arthritis-Related Pain via Direct Activation of Primary Sensory Neurons

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    The tachykinin hemokinin-1 (HK-1) is involved in immune cell development and inflammation, but little is known about its function in pain. It acts through the NK1 tachykinin receptor, but several effects are mediated by a yet unidentified target. Therefore, we investigated the role and mechanism of action of HK-1 in arthritis models of distinct mechanisms with special emphasis on pain. Arthritis was induced by i.p. K/BxN serum (passive transfer of inflammatory cytokines, autoantibodies), intra-articular mast cell tryptase or Complete Freund’s Adjuvant (CFA, active immunization) in wild type, HK-1- and NK1-deficient mice. Mechanical- and heat hyperalgesia determined by dynamic plantar esthesiometry and increasing temperature hot plate, respectively, swelling measured by plethysmometry or micrometry were significantly reduced in HK-1-deleted, but not NK1-deficient mice in all models. K/BxN serum-induced histopathological changes (day 14) were also decreased, but early myeloperoxidase activity detected by luminescent in vivo imaging increased in HK-1-deleted mice similarly to the CFA model. However, vasodilation and plasma protein extravasation determined by laser Speckle and fluorescent imaging, respectively, were not altered by HK-1 deficiency in any models. HK-1 induced Ca2+-influx in primary sensory neurons, which was also seen in NK1-deficient cells and after pertussis toxin-pretreatment, but not in extracellular Ca2+-free medium. These are the first results showing that HK-1 mediates arthritic pain and cellular, but not vascular inflammatory mechanisms, independently of NK1 activation. HK-1 activates primary sensory neurons presumably via Ca2+ channel-linked receptor. Identifying its target opens new directions to understand joint pain leading to novel therapeutic opportunities

    Investigation of Transient Receptor Ankyrin 1, Vanilloid 1 Ion Channels and Pituitary Adenylate Cyclase Polypeptide in the Uterus and Mammary Gland

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    The Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) are non-selective cation channels predominantly localized on capsaicin-sensitive peptidergic sensory neurons and mediate pain and inflammation. TRPV1 or „capsaicin receptor” is activated by noxious heat (>43°C), protons (pH>5.5), bradykinin, lipoxygenase products and anandamide produced during inflammation and tissue injury. TRPV1 is also expressed in the central nervous system (CNS) and on several non-neuronal cells in the skin, kidney, lung, testis, pancreas, spleen, cornea, and the uterus. TRPA1 is also activated by various chemical and physical stimuli, such as noxious cold (<17C°), allyl-isothiocyanate, cinnamaldehyde, as well as endogenous ligands like hydrogen peroxide, formaldehyde, methylglyoxal and acrolein produced during inflammation and tissue damage. The pathophysiological relevance of TRPA1 has been shown in inflammatory diseases of the respiratory, cardiovascular and gastrointestinal tracts. Similarly to TRPV1, functional TRPA1 was also described on enterochromaffin cells, synoviocytes, fibroblasts, melanocytes, pancreatic beta cells, epidermal keratinocytes, intestinal epithelial cells, and macrophages, as well as human endometrium cells besides sensory neurons. Although the physiological/pathophysiological relevance of non-neuronal TRP is unknown, a cross-talk has been proposed between non-neuronal and neuronal TRP channels. The expression of TRPV1 at protein level has been shown in the intact human endometrium at both neuronal and non-neuronal sites. Although the non-neuronal receptor expression was steady during the menstrual cycle, neuronal TRPV1 expression presumably has an estrogen-dependent regulation The consistent upregulation of TRPV1 in the peritoneal and endometrial tissues of women with chronic pelvic pain suggests its potential significance in various gynecological pain symptoms. Further research revealed increased TRPV1 expression at both neuronal and non-neuronal sites in the peritoneal endometriosis lesions and endometrioma. Despite these data on TRPV1 expression in the human endometrium and association with constant severe pelvic pain, there are no data about its expression in DIE. Furthermore, there is no information about TRPA1 expression in the human endometrium at all

    Expression and estrogen-dependent up-regulation of Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) ion channels in the rat endometrium

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    Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) cation channels localized predominantly on capsaicin-sensitive peptidergic sensory nerves play essential roles in pain, hyperalgesia and neurogenic inflammation. They are activated by a variety of noxious stimuli, chemical irritants and cold or heat, respectively. Besides sensory nerves, both receptors have been described on epithelial and immune cells. Estrogen-induced TRPV1 up-regulation in the human uterus suggests its potential involvement in pain during the reproductive cycle. Since there are no data regarding TRPA1 expression in the endometrium and little is known about TRPV1 regulation, we investigated estrogen- and progesterone-dependent alterations of these channels in the rat endometrium. Different groups of sexually premature 4-week-old and adult 4-month-old female rats were treated with subcutaneously implanted wax pellets containing synthetic estrogen analog diethylstilbestrol (DES, 100 µg), progesterone (4 mg) and their combination for 8 or 12 days, respectively. Ovariectomy was performed in separate groups of 4-month-old animals (n=5/group). TRPA1 and TRPV1 mRNA levels were measured in the endometrium layer with quantitative PCR, while the localization of the receptor proteins was determined with immunohistochemistry on paraffin-embedded uterus sections. Both TRPA1 and TRPV1 were detected in the rat endometrium at mRNA and protein levels as well, showing their remarkable local, non-neuronal expression. DES treatment resulted in a 5-fold and 7-fold significant up-regulation of TRPV1 mRNA in young and adult rats, respectively, which were absent if progesterone was added simultaneously. DES also induced significant elevation of TRPA1 mRNA in both groups. Progesterone by itself did not alter the levels of either channel in either group. In young rats, weak TRPV1 and A1 staining were observed in the epithelium, while in adult animals it was detected in the stroma and the glands with weak expression in the epithelium. Further investigations are in process to elucidate the functions of TRPA1 and TRPV1 in conditions related to pain and inflammation. SROP-4.2.2.A-11/1/KONV-2012-002

    Synthetic Diphenylacetylene-Based Retinoids Induce DNA Damage in Chinese Hamster Ovary Cells without Altering Viability

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    All-trans-retinoic acid (ATRA), the active metabolite of vitamin A, plays a pivotal role in cell differentiation, proliferation and embryonic development. It is an effective therapy for dermatological disorders and malignancies. ATRA is prone to isomerization and oxidation, which can affect its activity and selectivity. Novel diphenylacetylene-based ATRA analogues with increased stability can help to overcome these problems and may offer significant potential as therapeutics for a variety of cancers and neurodegenerative diseases, including amyotrophic lateral sclerosis. Here, we investigated the effects of these retinoids on cell viability and genotoxicity in the widely used model system of the rapidly proliferating Chinese hamster ovary cell line. DC360 is a fluorescent ATRA analogue and DC324 is a non-active derivative of DC360. EC23, DC525, DC540, DC645, and DC712 are promising analogues with increased bioactivity. The cytotoxic activity of the compounds was evaluated by ATP assay and DNA damage was tested by comet assay. No cytotoxicity was observed in the 10−6–10−5 M concentration range. All compounds induced DNA migration similar to ATRA, but DC324, DC360 and EC23 did so to a greater extent, particularly at higher concentrations. We believe that retinoid receptor-independent genotoxicity is a general characteristic of these compounds; however, further studies are needed to identify the molecular mechanisms and understand their complex biological functions

    Analysis of longevity in Holstein Friesian cattle using proteomic approaches

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    The aim of the present study was to determine marker proteins those are associated with functional longevity of dairy cattle. Holstein-Friesian cows were grouped based on their performance as follows: group 1) individuals with good longevity traits; group 2) short production life because of poor reproduction traits; group 3) short production life with low milk yield. Twelve individuals were sampled in each group, blood and milk samples were collected from cows. Blood samples were analysed with two dimensional polyacrylamide gel electrophoresis (2D PAGE), MALDI TOF/TOF and nanoLCMS/MS. The milk samples were analysed with MALDI TOF/TOF and nanoLC-MS/MS. Using the optimized gel based proteomic approach,we have succesfully separated 143 proteins in the group1, 139 proteins in the group2 and 136 proteins in the group3, but we could not find significant differences between groups in the expression pattern. Using MALDI TOF/TOF and nanoLC-IonTrap MS, we have found eleven protein sequences those were expressed only in the samples of good longevity group
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