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

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

Resolvin D1 and D2 inhibit transient receptor potential vanilloid 1 and ankyrin 1 ion channel activation on sensory neurons via lipid raft modification

Transient Receptor Potential Vanilloid 1 and Ankyrin 1 (TRPV1, TRPA1) cation channels are expressed in nociceptive primary sensory neurons and regulate nociceptor and inflammatory functions. Resolvins are endogenous lipid mediators. Resolvin D1 (RvD1) is described as a selective inhibitor of TRPA1-related postoperative and inflammatory pain in mice acting on the G protein-coupled receptor DRV1/GPR32. Resolvin D2 (RvD2) is a very potent TRPV1 and TRPA1 inhibitor in DRG neurons, and decreases inflammatory pain in mice acting on the GPR18 receptor, via TRPV1/TRPA1-independent mechanisms. We provided evidence that resolvins inhibited neuropeptide release from the stimulated sensory nerve terminals by TRPV1 and TRPA1 activators capsaicin (CAPS) and allyl-isothiocyanate (AITC), respectively. We showed that RvD1 and RvD2 in nanomolar concentrations significantly decreased TRPV1 and TRPA1 activation on sensory neurons by fluorescent calcium imaging and inhibited the CAPS-and AITC-evoked45Ca-uptake on TRPV1-and TRPA1-expressing CHO cells. Since CHO cells are unlikely to express resolvin receptors, resolvins are suggested to inhibit channel opening through surrounding lipid raft disruption. Here, we proved the ability of resolvins to alter the membrane polarity related to cholesterol composition by fluorescence spectroscopy. It is concluded that targeting lipid raft integrity can open novel peripheral analgesic opportunities by decreasing the activation of nociceptors. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

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

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

Somatostatin and Its Receptors in Myocardial Ischemia/Reperfusion Injury and Cardioprotection

Little is known about the role of the neuropeptide somatostatin (SST) in myocardial ischemia/reperfusion injury and cardioprotection. Here, we investigated the direct cardiocytoprotective effect of SST on ischemia/reperfusion injury in cardiomyocyte cultures, as well as the expression of SST and its receptors in pig and human heart tissues. SST induced a bell-shaped, concentration-dependent cardiocytoprotection in both adult rat primary cardiomyocytes and H9C2 cells subjected to simulated ischemia/ reperfusion injury. Furthermore, in a translational porcine closed-chest acute myocardial infarction model, ischemic preconditioning increased plasma SST-like immunoreactivity. Interestingly, SST expression was detectable at the protein, but not at the mRNA level in the pig left ventricles. SSTR1 and SSTR2, but not the other SST receptors, were detectable at the mRNA level by PCR and sequencing in the pig left ventricle. Moreover, remote ischemic conditioning upregulated SSTR1 mRNA. Similarly, SST expression was also detectable in healthy human interventricular septum samples at the protein level. Furthermore, SST-like immunoreactivity decreased in interventricular septum samples of patients with ischemic cardiomyopathy. SSTR1, SSTR2, and SSTR5 but not SST and the other SST receptors were detectable at the mRNA level by sequencing in healthy human left ventricles. In addition, in healthy human left ventricle samples, SSTR1 and SSTR2 mRNAs were expressed especially in vascular endothelial and some other cell types as detected by RNA Scope® in situ hybridization. This is the first demonstration that SST exerts a direct cardiocytoprotective effect against simulated ischemia/reperfusion injury. Moreover, SST is expressed in the heart tissue at the peptide level; however, it is likely to be of sensory neural origin since its mRNA is not detectable. SSTR1 and SSTR2 might be involved in the cardioprotective action of SST, but other mechanisms cannot be excluded

Characterization of Neurons Expressing the Novel Analgesic Drug Target Somatostatin Receptor 4 in Mouse and Human Brains

Somatostatin is an important mood and pain-regulating neuropeptide, which exerts analgesic, anti-inflammatory, and antidepressant effects via its Gi protein-coupled receptor subtype 4 (SST4) without endocrine actions. SST4 is suggested to be a unique novel drug target for chronic neuropathic pain, and depression, as a common comorbidity. However, its neuronal expression and cellular mechanism are poorly understood. Therefore, our goals were (i) to elucidate the expression pattern of Sstr4/SSTR4 mRNA, (ii) to characterize neurochemically, and (iii) electrophysiologically the Sstr4/SSTR4-expressing neuronal populations in the mouse and human brains. Here, we describe SST4 expression pattern in the nuclei of the mouse nociceptive and anti-nociceptive pathways as well as in human brain regions, and provide neurochemical and electrophysiological characterization of the SST4-expressing neurons. Intense or moderate SST4 expression was demonstrated predominantly in glutamatergic neurons in the major components of the pain matrix mostly also involved in mood regulation. The SST4 agonist J-2156 significantly decreased the firing rate of layer V pyramidal neurons by augmenting the depolarization-activated, non-inactivating K+ current (M-current) leading to remarkable inhibition. These are the first translational results explaining the mechanisms of action of SST4 agonists as novel analgesic and antidepressant candidates

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

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

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

Comparative proteome analysis of skeletal muscle between Merino and Tsigai lambs

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