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Despite campaigns and improvements in detection and treatment, lung cancer continues to increase worldwide and represents a major public health problem. One approach to treating patients suffering from lung cancer is to target surface receptors overexpressed on tumor cells, such as GPCR-family kinin receptors, and proteases that control tumor progression, such as kallikrein-related peptidases (KLKs). These proteases have been visualized in recent years due to their contribution to the progression of cancers, such as prostate and ovarian cancer, facilitating the invasive and metastatic capacity of tumor cells in these tissues. In fact, KLK3 is the specific prostate antigen, the only tissue-specific biomarker used to diagnose this malignancy. In lung cancer to date, evidence indicates that KLK5, KLK6, KLK8, KLK11, and KLK14 are the major peptidases regulated and involved in its progression. The expression levels of KLKs in this neoplasm are modulated by the secretome of the different cell types present in the tumor microenvironment, the cancer subtype and the tumor stage, among others. Considering the multiple functions of kinin receptors and KLKs, this review highlights their roles, even considering the SARS-CoV-2 effects. Since lung cancer is often diagnosed in advanced stages, our efforts should focus on early diagnosis, validating for example specific KLKs, especially in high-risk populations such as smokers and people exposed to carcinogenic fumes, oil fields, and contaminated workplaces, unexplored fields to investigate. Furthermore, their modulation could be considered as a promising approach in lung cancer therapeutics., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The epidermis is the outermost skin layer and is part of one of the largest organs in the body; it is supported by the dermis, a network of fibrils, blood vessels, pilosebaceous units, sweat glands, nerves, and cells. The skin as a whole is a protective shield against numerous noxious agents, including microorganisms and chemical and physical factors. These functions rely on the activity of multiple growth factors, peptide hormones, proteases, and specific signaling pathways that are triggered by the activation of distinct types of receptors sited in the cell membranes of the various cell types present in the skin. The human kallikrein family comprises a large group of 15 serine proteases synthesized and secreted by different types of epithelial cells throughout the body, including the skin. At this site, they initiate a proteolytic cascade that generates the active forms of the proteases, some of which regulate skin desquamation, activation of cytokines, and antimicrobial peptides. Kinin peptides are formed by the action of plasma and tissue kallikreins on kininogens, two plasma proteins produced in the liver and other organs. Although kinins are well known for their proinflammatory abilities, in the skin they are also considered important modulators of keratinocyte differentiation. In this review, we summarize the contributions of the kallikreins and kallikrein-related peptidases family and those of kinins and their receptors in skin homeostasis, with special emphasis on their pathophysiological role.

Ion channels are potentially exploitable as pharmacological targets to treat asthma. This study evaluated the role of KCa3.1 channels, encoded by Kcnn4 , in regulating the gene expression of mouse airway epithelium and the development of asthma traits. We used the ovalbumin (OVA) challenge as an asthma model in wild-type and Kcnn4 -/- mice, performed histological analysis, and measured serum IgE to evaluate asthma traits. We analyzed gene expression of isolated epithelial cells of trachea or bronchi using mRNA sequencing and gene ontology and performed Ussing chamber experiments in mouse trachea to evaluate anion secretion. Gene expression of epithelial cells from mouse airways differed between trachea and bronchi, indicating regional differences in the inflammatory and transepithelial transport properties of proximal and distal airways. We found that Kcnn4 silencing reduced mast cell numbers, mucus, and collagen in the airways, and reduced the amount of epithelial anion secretion in the OVA-challenged animals. In addition, gene expression was differentially modified in the trachea and bronchi, with Kcnn4 genetic silencing significantly altering the expression of genes involved in the TNF pathway, supporting the potential of KCa3.1 as a therapeutic target for asthma.

Sweet syndrome (SS) is an acute febrile neutrophilic dermatosis that is histologically characterized by an infiltration of the dermis by neutrophils. A 12-year-old adolescent female patient recently diagnosed with acute promyelocytic leukemia presented with fever and was hospitalized for antibiotic management after 22 days of being treated with a treatment protocol based on daunorubicin, all-trans retinoic acid (ATRA), and prophylaxis with dexamethasone, the patient developed erythematous skin lesions located mostly on the extremities. Lesions evolved into painful subcutaneous nodules, and one lesion evolved into a 2.5-cm blister with a purple and necrotic base. A skin biopsy was performed and showed neutrophilic dermatosis which confirmed the diagnosis of SS. The patient's clinical features complied with criteria for differentiation syndrome complicated by shock. Two days after ATRA was suspended, the patient presented resolution of the fever and skin lesions. SS is a rare neutrophilic dermatosis secondary to an innate immune disorder classified into four categories: classical (idiopathic), para-inflammatory, paraneoplastic or pregnancy-related. SS has been described in patients with acute myeloid leukemia in adults secondary to the use of drugs such as ATRA or as a part of a paraneoplastic syndrome. SS can occur exceptionally in children with myeloid leukemia secondary to the use of drugs such as ATRA., Competing Interests: The authors declare that they have no conflict of interest., (Copyright 2021, Ramirez et al.)

Estrogens and estrogen-like molecules can modify the biology of several cell types. Estrogen receptors alpha (ERα) and beta (ERβ) belong to the so-called classical family of estrogen receptors, while the G protein-coupled estrogen receptor 1 (GPER-1) represents a non-classical estrogen receptor mainly located in the plasma membrane. As estrogen receptors are ubiquitously distributed, they can modulate cell proliferation, differentiation, and survival in several tissues and organs, including the central nervous system (CNS). Estrogens can exert neuroprotective roles by acting as anti-oxidants, promoting DNA repair, inducing the expression of growth factors, and modulating cerebral blood flow. Additionally, estrogen-dependent signaling pathways are involved in regulating the balance between proliferation and differentiation of neural stem/progenitor cells (NSPCs), thus influencing neurogenic processes. Since several estrogen-based therapies are used nowadays and estrogen-like molecules, including phytoestrogens and xenoestrogens, are omnipresent in our environment, estrogen-dependent changes in cell biology and tissue homeostasis have gained attention in human health and disease. This article provides a comprehensive literature review on the current knowledge of estrogen and estrogen-like molecules and their impact on cell survival and neurodegeneration, as well as their role in NSPCs proliferation/differentiation balance and neurogenesis., Competing Interests: FB-B was employed by company Cells for Cells (Chile). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bustamante-Barrientos, Méndez-Ruette, Ortloff, Luz-Crawford, Rivera, Figueroa, Molina and Bátiz.)

GPER-1 is a novel membrane sited G protein-coupled estrogen receptor. Clinical studies have shown that patients suffering an estrogen receptor α (ERα)/GPER-1 positive, breast cancer have a lower survival rate than those who have developed ERα-positive/GPER-1 negative tumors. Moreover, absence of GPER-1 improves the prognosis of patients treated with tamoxifen, the most used selective estrogen receptor modulator to treat ERα-positive breast cancer. MCF-7 breast cancer cells were continuously treated with 1,000 nM tamoxifen for 7 days to investigate its effect on GPER-1 protein expression, cell proliferation and intracellular [Ca 2+ ] i mobilization, a key signaling pathway. Breast cancer cells continuously treated with tamoxifen, exhibited a robust [Ca 2+ ] i mobilization after stimulation with 1,000 nM tamoxifen, a response that was blunted by preincubation of cells with G15, a commercial GPER-1 antagonist. Continuously treated cells also displayed a high [Ca 2+ ] i mobilization in response to a commercial GPER-1 agonist (G1) and to estrogen, in a magnitude that doubled the response observed in untreated cells and was almost completely abolished by G15. Proliferation of cells continuously treated with tamoxifen and stimulated with 2,000 nM tamoxifen, was also higher than that observed in untreated cells in a degree that was approximately 90% attributable to GPER-1. Finally, prolonged tamoxifen treatment did not increase ERα expression, but did overexpress the kinin B1 receptor, another GPCR, which we have previously shown is highly expressed in breast tumors and increases proliferation of breast cancer cells. Although we cannot fully extrapolate the results obtained in vitro to the patients, our results shed some light on the occurrence of drug resistance in breast cancer patients who are ERα/GPER-1 positive, have been treated with tamoxifen and display low survival rate. Overexpression of kinin B1 receptor may explain the increased proliferative response observed in breast tumors under continuous treatment with tamoxifen., (Copyright © 2020 Molina, Bustamante, Ortloff, Ramos, Ehrenfeld and Figueroa.)

Kallikrein-related peptidases (KLKs) are a family of serine proteases that when dysregulated may contribute to neuroinflammation and neurodegeneration. In the present review article, we describe what is known about their physiological and pathological roles with an emphasis on KLK6 and KLK8, two KLKs that are highly expressed in the adult central nervous system (CNS). Altered expression and activity of KLK6 have been linked to brain physiology and the development of multiple sclerosis. On the other hand, altered levels of KLK6 in the brain and serum of people affected by Alzheimer's disease and Parkinson's disease have been documented, pointing out to its function in amyloid metabolism and development of synucleinopathies. People who have structural genetic variants of KLK8 can suffer mental illnesses such as intellectual and learning disabilities, seizures, and autism. Increased expression of KLK8 has also been implicated in schizophrenia, bipolar disorder, and depression. Also, we discuss the possible link that exists between KLKs activity and certain viral infections that can affect the nervous system. Although little is known about the exact mechanisms that mediate KLKs function and their participation in neuroinflammatory and neurodegenerative disorders will open a new field to develop novel therapies to modulate their levels and/or activity and their harmful effects on the CNS., (Copyright © 2020 Mella, Figueroa, Otth and Ehrenfeld.)

Kinins are proinflammatory peptides that are formed in the skin by the enzymatic action of tissue kallikrein (KLK1) on kininogens. Tissue kallikrein is produced by eccrine sweat glands and also by cells of the stratum granulosum and other skin appendages. Kinin formation may be favored during inflammatory skin disorders when plasma constituents, including kininogens, extravasate from venules and capillaries, which have increased permeability in response to the plethora of inflammatory mediators generated in the course of acute inflammation. By activating either kinin B1 or B2 receptors, kinins modulate keratinocyte differentiation, which relays on activation of several signaling systems that follows receptor stimulation. Participation of the kinin B1 receptor in wound healing is still a matter of controversy though some studies indicate that B1 receptor stimulation regulates keratinocyte migration by controlling metalloproteases 2 and 9 production and by improving wound closure in a mouse model. Development of more stable kinin B1 receptor agonists may be beneficial to modulate wound healing, especially if we take into account that the B1 receptor is up-regulated by inflammation and by cytokines generated in the inflamed microenvironment., (Copyright ©2020, Yale Journal of Biology and Medicine.)

Cutaneous squamous cell carcinoma (cSCC) is one of the most common types of malignant skin cancer in dogs, representing 3.9-10.4% of all canine skin tumours. Although the metastatic potential of cSCC is debated, it appears to mimic that observed in man. In man, predictive histopathological features for metastasis include tumour depth, lesions >5-6 mm in depth, and invasion of muscle, cartilage or bone. In dogs, some reports have focused on the clinical features and long-term progression of cSCC, but a gold standard treatment has not yet been developed. We explored the protein expression of kallikrein-related peptidase 5 (KLK5), an important modulator of skin homeostasis, in normal canine skin and in examples of cSCC. KLK5 was highly expressed in the upper stratum granulosum, stratum corneum, hair follicles and sweat glands, skin sites where human KLK5 has been shown to be involved in physiological processes including keratinocyte desquamation, antimicrobial defence, lipid permeability and pigmentation. In cSCC, tumour cells at the deep margin, as well as those in the centre of keratin pearls, displayed cytoplasmic expression of KLK5. Some of the KLK5 immunoreactive cells also expressed vimentin, suggesting that they may be undergoing epithelial-mesenchymal transition and therefore have a more invasive behaviour than those expressing only KLK5. KLK5 may be a novel molecular biomarker useful for predicting prognosis of cSSC in dogs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Endometrial epithelial cells play a key defensive role as part of the innate immune response of cow uterus. An association between risk of acquiring infectious diseases and increased levels of free fatty acids postpartum has been suggested, and the use of omega-3 fatty acids such as docosahexaenoic acid (DHA) has been proposed as a beneficial strategy to improve immunity and fertility. The goal of our study was to demonstrate the presence of free fatty acid (FFA)-1 and 4 receptors in endometrial cells and to investigate their role on DHA interference in lipopolysaccharide (LPS)-induced inflammatory endometrial activation. We demonstrated that the bovine endometrial (BEND) cells line and bovine endometrium express both FFA1 and FFA4 receptors. FFA1 and FFA4 receptors were localized in the epithelium lining the endometrial cavity and in endometrial glands whereas in BEND cells a characteristic cell membrane localization of both receptors was observed. DHA, a FFA4 natural agonist, increased intracellular calcium mobilization in BEND cells, but the FFA1 agonists oleic and linoleic acids did not increase this response. DHA-induced intracellular calcium mobilization was inhibited by the FFA4 and FFA1 antagonists AH7614 and GW1100, respectively. DHA significantly reduced LPS-induced prostaglandin E2 (PGE2) production, but none of the antagonists reduced the effect produced by DHA. On the contrary, linoleic acid increased LPS-induced PGE2 production. In conclusion, endometrial cells express FFA4 and FFA1 receptors, and DHA induces intracellular calcium release via FFA4 and FFA1 receptors. DHA reduces PGE2, but this response was not mediated by FFA4 or FFA1 receptors.

Estrogens generated within endocrine organs and the reproductive system act as ligands for at least three types of estrogen receptors. Estrogen receptors α (ERα) and β (ERβ) belong to the so-called classical family of estrogen receptors, whereas the G protein-coupled receptor GPR30, also known as GPER-1, has been described as a novel estrogen receptor sited in the cell membrane of target cells. Furthermore, these receptors are under stimulation of a family of exogenous estrogens, known as phytoestrogens, which are a diverse group of non-steroidal plant compounds derived from plant food consumed by humans and animals. Because phytoestrogens are omnipresent in our daily diet, they are becoming increasingly important in both human health and disease. Recent evidence indicates that in addition to classical estrogen receptors, phytoestrogens also activate GPER-1 a relevant observation since GPER-1 is involved in several physiopathological disorders and especially in estrogen-dependent diseases such as breast cancer.The first estrogen receptors discovered were the classical ERα and ERβ, but from an evolutionary point of view G protein-coupled receptors trace their origins in history to over a billion years ago suggesting that estrogen receptors like GPER-1 may have been the targets of choice for ancient phytoestrogens and/or estrogens.This review provides a comprehensive and systematic literature search on phytoestrogens and its relationship with classical estrogen receptors and GPER-1 including its role in breast cancer, an issue still under discussion., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In the human neutrophil, kallikrein-related peptidases (KLKs) have a significant functional relationship with the classical kinin system as a kinin B1 receptor agonist induces secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the medium. Secretion of KLK1, the kinin-forming enzyme, may perpetuate formation of kinin in the inflammatory milieu by hydrolyzing extravasated kininogens present in tissue edema. Secretion of KLKs into the inflammatory milieu, induced by kinins or other proinflammatory mediators, provides the human neutrophil with a wide range of molecular interactions to hydrolyze different cellular and extracellular matrix components, which may be of critical relevance in different mechanisms involving inflammation.

The kallikrein family comprises tissue kallikrein and 14 kallikrein-related peptidases (KLKs) recognized as a subgroup of secreted trypsin- or chymotrypsin-like serine proteases. KLKs are expressed in many cellular types where they regulate important physiological activities such as semen liquefaction, immune response, neural development, blood pressure, skin desquamation and tooth enamel formation. Tissue kallikrein, the oldest member and kinin-releasing enzyme, and KLK3/PSA, a tumor biomarker for prostate cancer are the most prominent components of the family. Additionally, other KLKs have shown an abnormal expression in neoplasia, particularly in breast cancer. Thus, increased levels of some KLKs may increase extracellular matrix degradation, invasion and metastasis; other KLKs modulate cell growth, survival and angiogenesis. On the contrary, KLKs can also inhibit angiogenesis and produce tumor suppression. However, there is a lack of knowledge on how KLKs are regulated in tumor microenvironment by molecules present at the site, namely cytokines, inflammatory mediators and growth factors. Little is known about the signaling pathways that control expression/secretion of KLKs in breast cancer, and further how activation of PAR receptors may contribute to functional activity in neoplasia. A better understanding of these molecular events will allow us to consider KLKs as relevant therapeutic targets for breast cancer.

Fatty and hydroxycarboxylic acids are one of the main intermediates of energy metabolism in ruminants and critical in the milk production of cattle. High production demands on a dairy farm can induce nutritional imbalances and metabolism disorders, which have been widely associated with the onset of sterile inflammatory processes and increased susceptibility to infections. The literature suggests that short-chain fatty acids (SCFA), long-chain fatty acids (LCFA) and hydroxycarboxylic acids are relevant modulators of the host innate inflammatory response. For instance, increased SCFA and lactate levels are associated with subacute ruminal acidosis (SARA) and the activation of pro-inflammatory processes mediated by diverse leukocyte and vascular endothelial cells. As such, free LCFA and the ketone body β-hydroxybutyrate are significantly increased in the plasma 1-2 weeks postpartum, coinciding with the time period in which cows are more susceptible to acquiring infectious diseases that the host innate immune system should actively oppose. Today, many of these pro-inflammatory responses can be related to the activation of specific G protein-coupled receptors, including GPR41/FFA3 and GPR43/FFA2 for SCFA; GPR40/FFA1 and GPR120/FFA4 for LCFA, GPR109A/HCA2 for ketone body β-hydroxybutyrate, and GPR81/HCA 1 for lactate, all expressed in different bovine tissues. The activation of these receptors modulates the release of intracellular granules [e.g., metalloproteinase-9 (MMP-9) and lactoferrin], radical oxygen species (ROS) production, chemotaxis, and the production of relevant pro-inflammatory mediators. The article aimed to review the role of natural ligands and receptors and the resulting impact on the host innate immune reaction of cattle and, further, to address the most recent evidence supporting a potential connection to metabolic disorders., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Introduction: Breast cancer is clinically classified as 'estrogen-positive' when at least 1% of cancer cells stain for the estrogen receptor alpha (ERα). However, recent research on both basic and clinical aspects of breast cancer suggests that GPER-1 (G protein-coupled estrogen receptor-1) may have an important role in breast cancer. Areas covered: This review provides a comprehensive and systematic literature search on GPER-1. We have focused on the role of GPER-1 in breast cancer and on resistance to endocrine therapy, an unsolved clinical issue still under discussion. Expert opinion: The discovery of GPER-1 as a novel estrogen receptor is unique and the signaling pathways activated by its stimulation, when compared to the classical nuclear ERα, indicate a potential role of GPER-1 in the genesis and mechanisms of drug resistance in breast cancer. Tumors expressing ERα represent the largest group of breast cancer patients indicating that more women eventually die from ERα-positive breast tumors than from other more malignant breast cancer subtypes such as HER2-positive and the triple negative groups. It is important to develop new strategies on endocrine therapy with regard to ERα and GPER-1 receptors to achieve innovative successful therapeutic tools.

The B1 bradykinin receptor (BDKRB1) is a component of the kinin cascade localized in the human skin. Some of the effects produced by stimulation of BDKRB1 depend on transactivation of epidermal growth factor receptor (EGFR), but the mechanisms involved in this process have not been clarified yet. The primary purpose of this study was to determine the effect of a BDKRB1 agonist on wound healing in a mouse model and the migration and secretion of metalloproteases 2 and 9 from human HaCaT keratinocytes and delineate the signalling pathways that triggered their secretion. Although stimulation of BDKRB1 induces weak chemotactic migration of keratinocytes and wound closure in an in vitro scratch-wound assay, the BDKRB1 agonist improved wound closure in a mouse model. BDKRB1 stimulation triggers synthesis and secretion of both metalloproteases, effects that depend on the activity of EGFR and subsequent phosphorylation of ERK1/2 and p38 mitogen-activated protein kinases and PI3K/Akt. In the mouse model, immunoreactivity for both gelatinases was concentrated around wound borders. EGFR transactivation by BDKRB1 agonist involves Src kinases family and ADAM17. In addition to extracellular matrix degradation, metalloproteases 2 and 9 regulate cell migration and differentiation, cell functions that are associated with the role of BDKRB1 in keratinocyte differentiation. Considering that BDKRB1 is up-regulated by inflammation and/or by cytokines that are abundant in the inflammatory milieu, more stable BDKRB1 agonists may be of therapeutic value to modulate wound healing., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The bradykinin type 2 receptor (B2R), main effector of the pleiotropic kallikrein-kinin system (KKS), has been localized in the key sites related to placentation in human, rat and guinea pig utero-placental units. The present study was directed to characterize the content, the cellular and subcellular localization of B2R in the villi and basal plate of placentas from normal and preeclamptic pregnancies by means of western blotting, immunohistochemistry and immunoelectron microscopy. The protein content of B2R was demonstrated in both placental zones. The villous placenta of normal and preeclamptic pregnancies expressed B2R in syncytiotrophoblast and fetal endothelium; the basal plate displayed B2R in extravillous trophoblasts and decidual cells. Lastly, immunogold electron microscopy revealed B2R in fetal endothelium, syncytiotrophoblast, extravillous cytotrophoblasts and decidual cells; in all cell types the receptor was mainly located in the cytosol and nucleus. The protein content of placental homogenates and the immunoreactivity in the different cells types did not differ between both study groups; however the abundance of nuclear immunogold B2R positive beads in extravillous trophoblasts was greater in the normal than in the preeclamptic placentas. The purpose of describing nuclear B2R in the utero-placental unit, and its increment in normal extravillous trophoblasts, is to stimulate the study of the functional pathways that may be relevant to understand the local role of the B2R in normal and preeclamptic gestation., (Copyright © 2016 International Society for the Study of Hypertension in Pregnancy. Published by Elsevier B.V. All rights reserved.)

Aim: Neutrophils are the first cells to arrive at sites of injury. Nevertheless, many inflammatory diseases are characterized by an uncontrolled infiltration and action of these cells. Cell migration depends on volume changes that are governed by ion channel activity, but potassium channels in neutrophil have not been clearly identified. We aim to test whether KCa3.1 participates in neutrophil migration and other relevant functions of the cell., Methods: Cytometer and confocal measurements to determine changes in cell volume were used. Cells isolated from human, mouse and horse were tested for KCa3.1-dependent chemotaxis. Chemokinetics, calcium handling and release of reactive oxygen species were measured to determine the role of KCa3.1 in those processes. A mouse model was used to test for neutrophil recruitment after acute lung injury in vivo., Results: We show for the first time that KCa3.1 is expressed in mammalian neutrophils. When the channel is inhibited by a pharmacological blocker or by genetic silencing, it profoundly affects cell volume regulation, and chemotactic and chemokinetic properties of the cells. We also demonstrated that pharmacological inhibition of KCa3.1 did not affect calcium entry or reactive oxygen species production in neutrophils. Using a mouse model of acute lung injury, we observed that Kca3.1(-/-) mice are significantly less effective at recruiting neutrophils into the site of inflammation., Conclusions: These results demonstrate that KCa3.1 channels are key actors in the migration capacity of neutrophils, and its inhibition did not affect other relevant cellular functions., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

The injured skin produces a number of mediators that directly or indirectly modulate cell chemotaxis, migration, proliferation, and angiogenesis. Components of the kinin pathway including the kinin B1 receptor (B1R) have been found to occur in the human skin, but information about its role on keratinocyte biology is still scarce. Our aim was to determine whether stimulation of B1R causes the secretion of IL-4 and/or VEGF from human keratinocytes and to evaluate the role of the B1R agonist Lys-des[Arg(9)]bradykinin and IL-4 on various stages of angiogenesis, such as cell migration, proliferation, and release of metalloproteases. By using ELISA and Western blotting, we showed that HaCaT keratinocytes stimulated with the B1R agonist release IL-4 and VEGF. Stimulation of B1R also caused transient c-JunN-terminal kinase phosphorylation and JunB nuclear translocation, transcription factor that regulates IL-4 expression. The 3D-angiogenesis assay, performed on spheroids of EA.hy923 endothelial cells embedded in a collagen matrix, showed that their cumulative sprout area increased significantly following stimulation with either IL-4 or B1R agonist. Furthermore, these ligands produced significant endothelial cell migration and release of metalloproteases 2 and 9, but did not increase endothelial cell proliferation as measured by 5-bromo-2'-deoxyuridine incorporation. Our results provide experimental evidence that establishes IL-4 and B1R agonist as important angiogenic factors of relevance for skin repair.

The presence of high protein levels in the glomerular filtrate plays an important role in renal fibrosis, a disorder that justifies the use of animal models of experimental proteinuria. Such models have proved useful as tools in the study of the pathogenesis of chronic, progressive renal disease. Since bradykinin and the kinin B2 receptor (B2R) belong to a renoprotective system with mechanisms still unclarified, we investigated its anti-fibrotic role in the in vivo rat model of overload proteinuria. Upon up-regulating the kinin system by a high potassium diet we observed reduction of tubulointerstitial fibrosis, decreased renal expression of α-smooth muscle actin (α-SMA) and vimentin, reduced Smad3 phosphorylation and increase of Smad7. These cellular and molecular effects were reversed by HOE-140, a specific B2R antagonist. In vitro experiments, performed on a cell line of proximal tubular epithelial cells, showed that high concentrations of albumin induced expression of mesenchymal biomarkers, in concomitance with increases in TGF-β1 mRNA and its functionally active peptide, TGF-β1. Stimulation of the tubule cells by bradykinin inhibited the albumin-induced changes, namely α-SMA and vimentin were reduced, and cytokeratin recovered together with increase in Smad7 levels and decrease in type II TGF-β1 receptor, TGF-β1 mRNA and its active fragment. The protective changes produced by bradykinin in vitro were blocked by HOE-140. The development of stable bradykinin analogues and/or up-regulation of the B2R signaling pathway may prove value in the management of chronic renal fibrosis in progressive proteinuric renal diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The family of kallikrein-related peptidases (KLKs) has been identified in a variety of immunolabeled human tissue sections, but no previous study has experimentally confirmed their presence in the human neutrophil. We have investigated the expression and bioregulation of particular KLKs in the human neutrophil and, in addition, examined whether stimulation by a kinin B(1) receptor (B1R) agonist or fMet-Leu-Phe (fMLP) induces their secretion. Western blot analysis of neutrophil homogenates indicated that the MM of the KLKs ranged from 27 to 50 kDa. RT-PCR showed that blood neutrophils expressed only KLK1, KLK4, KLK10, KLK13, KLK14 and KLK15 mRNAs, whereas the non-differentiated HL-60 cells expressed most of them, with exception of KLK3 and KLK7. Nevertheless, mRNAs for KLK2, KLK5, KLK6 and KLK9 that were previously undetectable appeared after challenging with a mixture of cytokines. Both kinin B(1)R agonist and fMLP induced secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the culture medium in similar amounts, whereas the B(1)R agonist caused the release of lower amounts of KLK2, KLK4 and KLK5. When secreted, the differing proteolytic activity of KLKs provides the human neutrophil with a multifunctional enzymatic capacity supporting a new dimension for its role in human disorders of diverse etiology., (© The Author(s) 2015.)

Kinins are pro-inflammatory peptides that mimic the cardinal features of inflammation. We examined the concept that expression levels of endothelial intercellular adhesion molecule-1 (ICAM-1) and neutrophil integrins Mac-1 and LFA-1 are modulated by the kinin B1 receptor (B1R) agonist, Lys-des[Arg(9)]bradykinin (LDBK). Stimulation of endothelial cells with LDBK increased the levels of ICAM-1 mRNA transcripts/protein, and also of E-selectin and platelet endothelial adhesion molecule-1. ICAM-1 levels increased in a magnitude comparable with that produced by TNF-α. This stimulatory effect was reduced when endothelial cells, which had been previously transfected with a B1R small interfering RNA, were stimulated with LDBK, under comparable conditions. Similarly, LDBK produced a significant increase in protein levels of LFA-1 and Mac-1 integrins in human neutrophils, an effect that was reversed by pretreatment of cells with 10 µg/ml cycloheximide or a B1R antagonist. Functional experiments performed with post-confluent monolayers of endothelial cells stimulated with LDBK and neutrophils primed with TNF-α, and vice versa, resulted in enhanced adhesiveness between both cells. Neutralizing Abs to ICAM-1 and Mac-1 reduced the adhesion between them. Our results indicate that kinin B1R is a novel modulator that promotes adhesion of leukocytes to endothelial cells, critically enhancing the movement of neutrophils from the circulation to sites of inflammation., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

The sera of patients with breast cancer have higher levels of des[Arg(9)]bradykinin, a kinin B1 receptor (B1R) agonist, than that from healthy individuals. Stimulation of breast cancer cells with the analog Lys-des[Arg(9)]bradykinin causes release of metalloproteinases-2 and -9 and increases cell proliferation. We examined the possibility that breast cancer cells, in addition to B1R, express the kinin-forming protease true tissue kallikrein (KLK1) and the endogenous proteins termed kininogens from which kinins are enzymatically released. Furthermore, we investigated whether stimulation of breast cancer cells with a B1R agonist would modify the cellular levels of KLK6, KLK10 and KLK11, three kallikrein-related peptidases with a still poorly-understood biological role in breast cancer. We found that breast cancer cells expressed KLK1 and kininogens, and that stimulation of estrogen-sensitive breast cancer cells with the B1R agonist produced down-regulation of KLK10 (a protease associated with growth suppression) but up-regulation of KLK11 and KLK6 (peptidases related to increased cell proliferation and invasiveness, respectively). Furthermore, we showed that the B1R agonist acts as a functional stimulus for the secretion of KLK1 and KLK6, an event relevant for kinin production and cell invasion, respectively., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Antineoplastic treatment with cisplatin is frequently complicated by nephrotoxicity. Although oxidative stress may be involved, the pathogenic mechanisms responsible for renal damage have not been completely clarified. In order to investigate the role of the renal kinin system in this condition, a group of rats was submitted to high potassium diet to stimulate the synthesis and excretion of tissue kallikrein 1 (rKLK1) previous to an intraperitoneal injection of 7 mg/kg cisplatin. A significant reduction in lipoperoxidation, evidenced by urinary excretion of malondialdehyde and renal immunostaining of hidroxy-nonenal, was accompanied by a decline in apoptosis. Coincident with these findings we observed a reduction in the expression of renal KIM-1 suggesting that renoprotection may be occurring. Stimulation or indemnity of the renal kinin system deserves to be evaluated as a complementary pharmacological measure to diminish cisplatin nephrotoxicity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Mammalian target of rapamycin (mTOR) complex is a key regulator of autophagy, cell growth and proliferation. Here, we studied the effects of arginine vasopressin (AVP) on mTOR activation in vascular smooth muscle cells cultured in high glucose concentration. AVP induced the mTOR phosphorylation in A-10 cells grown in high glucose, in contrast to cells cultured in normal glucose; wherein, only basal phosphorylation was observed. The AVP-induced mTOR phosphorylation was inhibited by a PI3K inhibitor. Moreover, the AVP-induced mTOR activation inhibited autophagy and increased thymidine incorporation in cells grown in high glucose. This increase was abolished by rapamycin which inhibits the mTORC1 complex formation. Our results suggest that AVP stimulates mTOR phosphorylation by activating the PI3K/Akt signaling pathway and, subsequently, inhibits autophagy and raises cell proliferation in A-10 cells maintained in high glucose concentration., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The albumin overload model induces proteinuria and tubulointersitial damage, followed by hypertension when rats are exposed to a hypersodic diet. To understand the effect of kinin system stimulation on salt-sensitive hypertension and to explore its potential renoprotective effects, the model was induced in Sprague-Dawley rats that had previously received a high-potassium diet to enhance activity of the kinin pathway, followed with/without administration of icatibant to block the kinin B₂ receptor (B₂R). A disease control group received albumin but not potassium or icatibant, and all groups were exposed to a hypersodic diet to induce salt-sensitive hypertension. Potassium treatment increased the synthesis and excretion of tissue kallikrein (Klk1/rKLK1) accompanied by a significant reduction in blood pressure and renal fibrosis and with downregulation of renal transforming growth factor-β (TGF-β) mRNA and protein compared with rats that did not receive potassium. Participation of the B₂R was evidenced by the fact that all beneficial effects were lost in the presence of the B₂R antagonist. In vitro experiments using the HK-2 proximal tubule cell line showed that treatment of tubular cells with 10 nM bradykinin reduced the epithelial-mesenchymal transdifferentiation and albumin-induced production of TGF-β, and the effects produced by bradykinin were prevented by pretreatment with the B₂R antagonist. These experiments support not only the pathogenic role of the kinin pathway in salt sensitivity but also sustain its role as a renoprotective, antifibrotic paracrine system that modulates renal levels of TGF-β.

Introduction: Kinins are bioactive peptide hormones that exert biological effects by activating two types of G protein-coupled receptors namely, B(1) (B(1)R) and B(2) (B(2)R). These modulate normal physiological cellular functions, inflammatory disorders and carcinogenesis. New and novel kinin receptor antagonists have been synthesized and their efficacy evaluated., Areas Covered: The authors provide a comprehensive review on the cellular and molecular biology of kinins and their receptors is delineated along with evolution and discovery of selective peptide and non-peptide antagonists. The authors describe the in vitro and in vivo methods used to understand the relative functional roles of B(1)R and B(2)R in physiology and pathohysiology. Furthermore, the authors translate the evaluation of kinin antagonists in selected preclinical models and associated clinical indications. Literature was surveyed from original publications, standard sources, SciFinder, patent applications and clinical trials., Expert Opinion: The authors suggest that several key areas of functional biology need consideration, namely: re-evaluation, particularly in vivo, of the mechanism of action and relative functional roles of the B(1)R and B(2)R in physiology and acute and chronic disease in animals and man; need for improved animal models with increased use of humanized and human systems; development of fluorescent probes for use in vivo in animals and man using advanced imaging techniques; combination of kinin receptor antagonists and traditional chemotherapy for various cancers.

Aim: To determine the expression of HER2 and bradykinin B(1) receptors (B(1)R) in the two pathogenic models of gallbladder cancer: the metaplasia-dysplasia-carcinoma and the adenoma-carcinoma pathways., Methods: Receptor proteins were visualized by immunohistochemistry on 5-μm sections of paraffin-embedded tissue. Expression of both receptors was studied in biopsy samples from 92 patients (6 males and 86 females; age ranging from 28 to 86 years, mean 56 years). High HER2 expression in specimens was additionally investigated by fluorescence in situ hybridization. Cell proliferation in each sample was assessed by using the Ki-67 proliferation marker., Results: HER2 receptor protein was absent in adenomas and in normal gallbladder epithelium. On the contrary, there was intense staining for HER2 on the basolateral membrane of epithelial cells of intestinal metaplasia (22/24; 91.7%) and carcinoma in situ (9/10; 90%), the lesions that displayed a significantly high proliferation index. Protein up-regulation of HER2 in the epithelium with metaplasia or carcinoma in situ was not accompanied by HER2 gene amplification. A similar result was observed in invasive carcinomas (0/12). The B(1)R distribution pattern mirrored that of HER2 except that B(1)R was additionally observed in the adenomas. The B(1)R appeared either as cytoplasmic dots or labeling on the apical cell membrane of the cells composing the epithelia with intestinal metaplasia (24/24; 100%) and carcinoma in situ (10/10; 100%) and in the epithelial cells of adenomas. In contrast, both HER2 (4/12; 33%) and B(1)R (1/12; 8.3%) showed a low expression in invasive gallbladder carcinomas., Conclusion: The up-regulation of HER2 and B(1)R in precursor lesions of gallbladder carcinoma suggests cross-talk between these two receptors that may be of importance in the modulation of cell proliferation in gallbladder carcinogenesis.

Introduction: Biological fluids of cancer patients contain increased levels of kinins. Activation of kinin B1 and B2 receptors expressed on cancer cells produce an increase in cell proliferation, migration of tumor cells and release of MMPs, which are cellular and molecular events of primary importance for tumor growth. The effects of kinins on tumor cells may be amplified by stimulation of kinin receptors expressed on other cells, within the tumor microenvironment, which may in turn increase tumor growth., Areas Covered: This review provides a comprehensive discourse on kinins and their receptors in human neoplasia. Concepts that view kinin receptors as targets for human cancer are explored, whilst the molecular basis by which the new dimerized kinin receptor antagonists produce arrest of cell proliferation and apoptosis of cancer cells is also examined. Finally, the role of kinin receptor antagonists as therapeutic tools against human neoplasia is analyzed., Expert Opinion: At the present time the available potent, dimerized kinin peptide antagonists, are either specific for B1 or B2 receptors, or are effective on both receptor types. The novel approach of using kinin receptor antagonists either alone or in combination therapy will play a definitive role in the treatment of cancer.

The V2 receptor gene encodes two receptor variants by alternative splicing, the canonical V2 receptor (V2a receptor) and V2b. The V2b variant has an amino acid sequence identical to that of the V2a receptor up to the sixth transmembrane domain, but the V2b sequences corresponding to the putative seventh transmembrane domain and the carboxyl terminus are different from those of the V2a receptor. Here we investigate the topology and subcellular distribution of the V2b variant. We found that, in contrast to the V2a receptor, the V2b adopted two topologies: one with six transmembrane segments with the C-terminus on the extracellular side of the membrane and another with seven transmembrane segments with the C-terminus on the intracellular side, similar to typical G-protein-coupled receptors. Furthermore, we observed that both topological isoforms oligomerized with the V2a canonical receptor. Unlike the V2a receptor, V2b did not move to the plasma membrane, but it is retained in the ER--Golgi compartments. These findings indicate that the C-terminal sequence beyond the sixth transmembrane of the V2a is required for the stabilization of the seven-transmembrane topology of the receptor and is also essential for the trafficking of the receptor to the plasma membrane.

The kinin B(1) receptor (B(1)R) agonist Lys-des[Arg(9)]-bradykinin (LDBK) increases proliferation of estrogen-sensitive breast cancer cells by a process involving activation of the epidermal growth factor receptor (EGFR) and downstream signaling via the ERK1/2 mitogen-activated protein kinase pathway. Here, we investigated whether B(1)R stimulation induced release of the extracellular matrix metalloproteases MMP-2 and MMP-9 via ERK-dependent pathway in both estrogen-sensitive MCF-7 and -insensitive MDA-MB-231 breast cancer cells. Cells were stimulated with 1-100nM of the B(1)R agonist for variable time-points. Western blotting and gelatin zymography were used to evaluate the presence of MMP-2 and MMP-9 in the extracellular medium. Stimulation of B(1)R with as little as 1 nM LDBK induced the accumulation of these metalloproteases in the medium within 5-30min of stimulation. In parallel, immunocytochemistry revealed that metalloprotease levels in the breast cancer cells declined after stimulation. This effect was blocked either by pre-treating the cells with a B(1)R antagonist or by transfecting with B(1)R-specific siRNA. Activation of the ERK1/2 pathway and EGFR transactivation was required for release of metalloproteases because both the MEK1 inhibitor, PD98059, and AG1478, an inhibitor of the EGFR-tyrosine kinase activity, blocked this event. The importance of EGFR-dependent signaling was additionally confirmed since transfection of cells with the dominant negative EGFR mutant HERCD533 blocked the release of metalloproteases. Thus, activation of B(1)R is likely to enhance breast cancer cells invasiveness by releasing enzymes that degrade the extracellular matrix and thereby favor metastasis., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Kinin peptides exert multiple biological effects by binding to two types of G protein-coupled receptors known as B(1) (B(1)R) and B(2) receptors. Expression of the B(1)R in human breast cancer was recently reported, but up to now the consequences of its stimulation are unknown. Our aims were (1) to investigate the capacity of B(1)R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B(1)R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B(1)R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B(1)R that once stimulated triggered cell proliferation at nanomolar concentrations of the ligand. Inhibitor studies suggested that the proliferative effects depend on the activity of epidermal growth factor receptor and subsequent ERK1/2 mitogen-activated protein kinases phosphorylation. B(1)R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B(1)R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B(1)R may be a valuable alternative for the treatment of breast cancer.

During neutrophil activation and degranulation, MMP-9 and MPO are released into the extracellular space to propagate inflammatory disorders. As kinin peptides are major participants in acute inflammatory responses, and the G-protein-coupled B(1)R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP-9 and MPO by the B(1)R agonist LDBK and determined the signaling pathways that may regulate this cellular effect. Cytochalasin-treated and -untreated neutrophils were suspended in HBSS and stimulated with a range concentration of LDBK for 5 min. Zymography and Western blotting revealed that LDBK induced the release of MMP-9 and MPO. The use of specific signaling transduction inhibitors showed that release of MMP-9 depended on ERK1/2 and p38 MAPKs, whereas release of MPO involved only the p38 cascade. Inhibition of the key steps in these pathways showed that the release of both enzymes depended on PKC and PI3K. Stimulation of neutrophils with LDBK produced phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by B(1)R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B(1)R. Our results demonstrate that in human neutrophils, activation of kinin B(1)R by LDBK initiates separate signaling cascades that trigger the release of MMP-9 and MPO from tertiary and primary granules, respectively, suggesting that the B(1)R plays a pivotal role in inflammatory disorders.

Eosinophils contribute to asthmatic airway inflammation by releasing cysteinyl leukotrienes (cysLT) and other inflammatory mediators, and bradykinin (BK) induces bronchoconstriction in asthmatic patients. The aims of this study were to investigate kinin receptor expression on eosinophils of asthmatic and healthy subjects and to assess the effects of kinin stimulation on eosinophils, which were isolated from peripheral blood of asthmatic (n=27) and healthy subjects (n=14). Kinin B(1) and B(2) receptors (B(1)R and B(2)R, respectively) and mRNA expression were investigated by quantitative confocal microscopy, flow cytometry, and RT-PCR. Intracellular Ca(2+) was assessed by live-cell fluorescence confocal microscopy. Production of cysLT and eosinophil migration in response to BK and Lys-des[Arg(9)]-BK were assessed. Eosinophils expressed kinin B(1)R and B(2)R mRNA and proteins. Quantitative immunofluorescence analysis indicated that expression of B(1)R and B(2)R proteins was significantly greater in eosinophils of asthmatic patients compared with those of nonasthmatic subjects. However, kinin B(1)R and B(2)R mRNA expression did not differ significantly between these groups. Expression of kinin B(1)R and mRNA was decreased in patients using high doses of inhaled corticosteroids and in eosinophils treated with a corticosteroid in vitro. Kinin B(1) and B(2) agonists up-regulated expression of their respective receptors but did not increase intracellular Ca(2+) or the production of cysLT or enhance eosinophil migration significantly. Up-regulation of kinin receptor expression in eosinophils of asthmatic patients may be a consequence of inflammation, whereby enhanced release of kinin peptides has a positive-feedback effect on kinin receptor expression. Importantly, anti-inflammatory corticosteroids down-regulated the expression of the kinin B(1)R.

The importance of neutrophils in human disease such as rheumatoid arthritis, asthma, adult respiratory distress syndrome, and COPD has prompted the search for drugs capable to slow down neutrophil-dependent inflammation, without interference with innate immune responses. In this review, we summarize new potential drugs targets against neutrophils mediated inflammatory responses.

Vitamin C is reabsorbed from the renal lumen by one isoform of sodium-vitamin C co-transporters that mediate high affinity sodium-dependent L-ascorbic acid transport. Sodium-vitamin C cotransporter-1 mRNA has been detected in intestine and liver and the S3 segment of the renal proximal tubule. Here, we found that its distribution was broader and all three proximal tubule segments of mouse and human expressed the transporter but the S3 segment had the highest expression. Sodium-vitamin C co-transporter-1 expression was also found in the renal epithelial-derived LLC-PK1 cell line. Ascorbic acid transport in these cells was regulated by a single kinetic component that depended on the sodium concentration, pH and temperature. Reducing ascorbate concentration increased the apical expression of the transporter suggesting the presence of a feedback system for regulation of transporter abundance at the luminal membrane.

Objective: To evaluate the effects of methadone, administered alone or in combination with acepromazine or xylazine, on sedation and on physiologic values in dogs., Study Design: Randomized cross-over design., Animals: Six adult healthy mixed-breed dogs weighing 13.5 +/- 4.9 kg., Methods: Dogs were injected intramuscularly with physiologic saline (Control), or methadone (0.5mg kg(-1)) or acepromazine (0.1 mg kg(-1)) or xylazine (1.0 mg kg(-1)), or acepromazine (0.05 mg kg(-1)) plus methadone (0.5 mg kg(-1)) or xylazine (0.5 mg kg(-1)) plus methadone (0.5 mg kg(-1)) in a randomized cross-over design, with at least 1-week intervals. Sedation, pulse rate, indirect systolic arterial pressure, respiratory rate (RR), body temperature and pedal withdrawal reflex were evaluated before and at 15-minute intervals for 90 minutes after treatment., Results: Sedation was greater in dogs receiving xylazine alone, xylazine plus methadone and acepromazine plus methadone. Peak sedative effect occurred within 30 minutes of treatment administration. Pulse rate was lower in dogs that received xylazine either alone or with methadone during most of the study. Systolic arterial pressure decreased only in dogs receiving acepromazine alone. When methadone was administered alone, RR was higher than in other treatments during most of the study and a high prevalence of panting was observed. In all treatments body temperature decreased, this effect being more pronounced in dogs receiving methadone alone or in combination with acepromazine. Pedal withdrawal reflex was absent in four dogs receiving methadone plus xylazine but not in any dog in the remaining treatments., Conclusions and Clinical Relevance: Methadone alone produces mild sedation and a high prevalence of panting. Greater sedation was achieved when methadone was used in combination with acepromazine or xylazine. The combination xylazine-methadone appears to result in better analgesia than xylazine administered alone. Both combinations of methadone/sedative were considered effective for premedication in dogs.

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.

Background: Keratinocyte life span is modulated by receptors that control proliferation and differentiation, key processes during cutaneous tissue repair. The kinin B(1) receptor (B(1)R) has been reported in normal and pathological human skin, but so far there is no information about its role in keratinocyte biology., Objectives: To determine the consequence of kinin B(1)R stimulation on tyrosine phosphorylation, a key signalling mechanism involved in keratinocyte proliferation and differentiation., Methods: Subconfluent primary cultures of human keratinocytes were used to investigate tyrosine phosphorylation, epidermal growth factor receptor (EGFR) transactivation, cell proliferation and keratinocyte differentiation. Cell proliferation was assessed by measuring bromodeoxyuridine incorporation whereas assessment of cell differentiation was based on the expression of filaggrin, cytokeratin 10 (CK10) and involucrin., Results: The major proteins phosphorylated, after B(1)R stimulation, were of molecular mass 170, 125, 89 and 70 kDa. The 170- and 125-kDa proteins were identified as EGFR and p125(FAK), respectively. Phosphorylation was greatly reduced by GF109203X and by overexposure of keratinocytes to phorbol 12-myristate 13-acetate, indicating the participation of protein kinase C. B(1)R stimulation did not increase [Ca(2+)]i, but triggered EGFR transactivation, an event that involved phosphorylation of Tyr(845), Tyr(992) and Tyr(1068) of EGFR. B(1)R stimulation did not elicit keratinocyte proliferation, but triggered cell differentiation, visualized as an increase of filaggrin, CK10 and involucrin. Blockade of EGFR tyrosine kinase by AG1478, before B(1)R stimulation, produced an additional increase in filaggrin expression., Conclusions: The kinin B(1)R may contribute to keratinocyte differentiation and migration by triggering specific tyrosine signalling pathways or by interacting with the ErbB receptor family.

Activation of V(1a) receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. We found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V(1a) receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and beta-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways.

Plasma prekallikrein (PPK) is synthesised in hepatocytes and secreted into the blood, where it participates in the surface-dependent activation of blood coagulation, fibrinolysis, kinin generation and inflammation. Recently we demonstrated by quantitative RT-PCR that the human PPK gene is transcribed not only in the liver, but also in various non-hepatic human tissues at significant levels. However, up to now no reliable information is available concerning protein synthesis in the corresponding human tissues. Here we demonstrate by immunohistochemical studies that PPK or plasma kallikrein (PK) is localised in cells of different embryologically derived human tissues. In the human nephron, single cells of the distal tubules stained intensely, while the cytoplasm of cells forming proximal tubules and collecting ducts stained uniformly. PPK/PK was localised in hepatic epithelial cells of the liver, in cells of the pancreatic islet of Langerhans, in the interstitial Leydig cells of the testes, in the follicular and thecal granulosa cells of the ovary, and in the parotid gland, oesophagus, skin, respiratory tract, prostate and breast. We conclude that the cellular localisation of PPK/PK in multiple different progenitor-derived cells indicates specific cellular functions of this enzyme, in addition to its known function in the blood.

Intestinal fluid secretion is driven by apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR)-mediated efflux of Cl- that is concentrated in cells by basolateral Na(+)-K(+)-2Cl- cotransporters (NKCC1). An absolute requirement for Cl- efflux is the parallel activation of K(+) channels which maintain a membrane potential that sustains apical anion secretion. Both cAMP and Ca(2+) are intracellular signals for intestinal Cl- secretion. The K(+) channel involved in cAMP-dependent secretion has been identified as the KCNQ1-KCNE3 complex, but the identity of the K(+) channel driving Ca(2+)-activated Cl- secretion is controversial. We have now used a Kcnn4 null mouse to show that the intermediate conductance IK1 K(+) channel is necessary and sufficient to support Ca(2+)-dependent Cl- secretion in large and small intestine. Ussing chambers were used to monitor transepithelial potential, resistance and equivalent short-circuit current in colon and jejunum from control and Kcnn4 null mice. Na(+), K(+) and water content of stools was also measured. Distal colon and small intestinal epithelia from Kcnn4 null mice had normal cAMP-dependent Cl- secretory responses. In contrast, they completely lacked Cl- secretion in response to Ca(2+)-mobilizing agonists. Ca(2+)-activated electrogenic K(+) secretion was increased in colon epithelium of mice deficient in the IK1 channel. Na(+) and water content of stools was diminished in IK1-null animals. The use of Kcnn4 null mice has allowed us to demonstrate that IK1 K(+) channels are solely responsible for driving intestinal Ca(2+)-activated Cl- secretion. The absence of this channel leads to a marked reduction in water content in the stools, probably as a consequence of decreased electrolyte and water secretion.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.