Carlos B. González

Kinin B1 receptor activation turns on exocytosis of matrix metalloprotease-9 and myeloperoxidase in human neutrophils: involvement of mitogen-activated protein kinase family

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 B1R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP‐9 and MPO by the B1R 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 B1R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B1R. Our results demonstrate that in human neutrophils, activation of kinin B1R by LDBK initiates separate signaling cascades that trigger the release of MMP‐9 and MPO from tertiary and primary granules, respectively, suggesting that the B1R plays a pivotal role in inflammatory disorders.

Stimulation of the bradykinin B1 receptor induces the proliferation of estrogen-sensitive breast cancer cells and activates the ERK1/2 signaling pathway

Kinin peptides exert multiple biological effects by binding to two types of G protein-coupled receptors known as B1 (B1R) and B2 receptors. Expression of the B1R 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 B1R to trigger cell proliferation in breast cancer cells, (2) to explore some of the downstream events occurring after B1R stimulation that may be linked to cell proliferation, and (3) to determine whether human breast tumors express potentially active B1R assessed by the binding of a radiolabeled agonist. Breast cancer cells expressed both the mRNA and the immunoreactive protein of B1R 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. B1R binding sites, were detected in 3/4 fibroadenomas, in 4/4 ductal carcinomas in situ and in 11/13 invasive ductal carcinomas. The B1R-epidermal growth factor receptor crosstalk may be a key interaction that maintains tumor growth, and antagonism of B1R may be a valuable alternative for the treatment of breast cancer.

Activation of kinin B1 receptor increases the release of metalloproteases-2 and -9 from both estrogen-sensitive and -insensitive breast cancer cells.

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.

Stimulated human neutrophils form biologically active kinin peptides from high and low molecular weight kininogens

Human neutrophils play a pivotal role in acute inflammation. However, their capacity to generate bioactive kinin peptides has not been established as yet. We have examined the ability of neutrophil enzymes to release biologically active kinins in vitro from purified human H‐ and L‐kininogens. Neutrophils isolated from human blood were stimulated with f‐Met‐Leu‐Phe, thrombin, or human immunoglobulin G adsorbed to silica particles. Supernatants were incubated with iodinated kininogens, and polyacrylamide gel electrophoresis analyzed aliquots taken after a range of incubation times. A time‐course analysis demonstrated that supernatants from stimulated neutrophils caused a rapid hydrolysis of both substrates, resulting in an accumulation of fragments ranging from 20 to less than 10 kDa. Radioimmunoassay (RIA) revealed that all supernatants were able to generate kinins in vitro. High‐performance liquid chromatography of the generated peptides indicated that they had a retention time similar to that of bradykinin and Met‐Lys‐bradykinin, clearly recognized as kinin peptides when the corresponding fractions were tested by RIA. The kinin‐immunoreactive fractions produced lowering of blood pressure and a dramatic increase in venular permeability. Biological activity of the neutrophil‐generated kinins was completely abolished by the B2 receptor antagonist HOE140, indicating that over the time‐course of the experiments, only kinin B2 agonists appeared to have been generated and that cellular actions of these were mediated by kinin B2 receptors. Together, our results demonstrate that human neutrophil proteases can release kinins from both plasma kininogens, suggesting that these peptides may participate actively during acute inflammation.

The pituitary cleft of the rat: An electron microscopic study

The epithelium of the pituitary cleft and its colloid have been studied at the ultrastructural level in normal, adrenalectomized, castrated, dehydrated and lactating adult rats. Both control and experimental animals showed cells in different stages of degradation inside the cleft and mixed with the colloid. These exfoliated cells seems to have originated from the clefts epithelium of the adjacent granular cells. In adrenalectomized and castrated rats the rostral epithelium of the cleft had large intercellular spaces that in some instances appeared to open directly into the cleft. Occasionally, the adenohypophysial canaliculi of the adrenalectomized rats were also seen connecting with the cleft. In lactating and castrated animals dark cells appeared more frequently in the rostral and caudal epithelium of the cleft. The results obtained suggest that the colloids is originated at least in part by involuted pituitary cells. There is also evidence that the follicular cavities of the Pars distalis and the cleft are continuous structures.

Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

Alzheimers disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) peptide. We have previously shown that the compound tetrahydrohyperforin (IDN5706) prevents accumulation of Aβ species in an in vivo model of AD, however the mechanism that explains this reduction is not well understood. We show herein that IDN5706 decreases the levels of ER degradation enhancer, mannosidase alpha-like 1 (EDEM1), a key chaperone related to endoplasmic-reticulum-associated degradation (ERAD). Moreover, we observed that low levels of EDEM1 correlated with a strong activation of autophagy, suggesting a crosstalk between these two pathways. We observed that IDN5706 perturbs the glycosylation and proteolytic processing of the amyloid precursor protein (APP), resulting in the accumulation of immature APP (iAPP) in the endoplasmic reticulum. To investigate the contribution of autophagy, we tested the effect of IDN5706 in Atg5-depleted cells. We found that depletion of Atg5 enhanced the accumulation of iAPP in response to IDN5706 by slowing down its degradation. Our findings reveal that IDN5706 promotes degradation of iAPP via the activation of Atg5-dependent autophagy, shedding light on the mechanism that may contribute to the reduction of Aβ production in vivo.

Ultrastructure and immunocytochemistry of neurons in the supraoptic and paraventricular nuclei of the lizard Liolaemus cyanogaster

SummaryThe supraoptic (SON) and paraventricular (PVN) nuclei of the lizard Liolaemus cyanogaster c. were studied by use of histochemical, immunocyto-chemical and electron microscopic methods. The immunofluorescence staining for neurophysin was applied to methacrylate-embedded material before and after treatment of the sections with urea and trypsin. Pseudoisocyanine was applied to sections previously used for immunocytochemistry. The ultrastructural study showed that the SON and PVN neurons possess neurosecretory granules (nsg), distributed throughout the perikaryon, and large (2 to 12 μm) electron-dense droplets located within dilatations of the cisternae of the rough endoplasmic reticulum. Whereas the perikaryon (nsg) and the secretory droplets are stainable with pseudoisocyanine, only the former displays immunoreactivity for neurophysin. However, after treating the sections with urea and trypsin, the same secretory droplets become immunoreactive. It is suggested that the secretory droplets are sites of storage for the precursor of neurophysin, and that the tryptic digestion either triggers its conversion into neurophysin or exposes its immunoreactive sites. Based on the ultrastructure and the histochemical behavior of the secretory droplets, it is also postulated that they contain, in addition to peptides, a glycoprotein component.

Expression and bioregulation of the kallikrein-related peptidases family in the human neutrophil

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 B1 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 B1R agonist and fMLP induced secretion of KLK1, KLK6, KLK10, KLK13 and KLK14 into the culture medium in similar amounts, whereas the B1R 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.

SVCT2 transporter expression is post‐natally induced in cortical neurons and its function is regulated by its short isoform

Different studies have demonstrated the importance of micronutrients, such as vitamins, for normal adult brain function and development. Vitamin C is not synthesized in the brain, but high levels are detected in this organ because of the existence of specific uptake mechanisms, which concentrate ascorbic acid from the bloodstream to the cerebrospinal fluid and then into neurons and glial cells. Two different isoforms of sodium–vitamin C cotransporters (SVCT1 and SVCT2) have been cloned. SVCT2 expression has been observed in the adult hippocampus and cortical neurons by in situ hybridization. In addition, the localization of SVCT2 in the rat fetal brain has been studied by immunohistochemistry and in situ hybridization, demonstrating that SVCT2 is highly expressed in the ventricular and subventricular areas of the brain cortex. However, there are currently no immunohistochemical data regarding SVCT2 expression and function in the post‐natal brain. Therefore, we analyzed SVCT2 expression in the developing brain cortex of mice, and demonstrated an increase in SVCT2 mRNA in mice at 1–15 days of age. The expression of a short isoform, SVCT2sh, was also detected within the same period. SVCT2 expression was concentrated in neurons within the inner layer of the brain cortex. Both SVCT2 isoforms were coexpressed in N2a cells to obtain functional data. Fluorescence resonance energy transfer analysis revealed a molecular interaction between SVCT2wt and SVCT2sh. Finally, differences in transport ratios suggested that SVCT2sh expression inhibited ascorbic acid uptake in N2a cells when both isoforms were coexpressed.

Identification and distribution of the cell types in the pituitary gland of Austromenidia laticlavia (Teleostei, Atherinidae).

By using antisera against human pituitary hormones in immunocytochemistry in combination with classical cytochemical techniques, we have been able to identify the different cell types in the adenohypophysis of the Austromenidia laticlavia and to determine their location. Antisera against prolactin and growth hormones did not stain cells in the pituitary of Austromenidia, whereas antisera against beta-endorphin, LH, and beta-TSH selectively cross-reacted with cells which have a specific location within the adenohypophysis. The beta-endorphin antiserum stained the periodic acid-Schiff (PAS)-negative cells in the pars intermedia and also, though faintly, the PAS-negative cells in the internal border of the rostral pars distalis (RPD). Human beta-TSH antiserum showed a discrete population of small PAS-positive cells in the proximal pars distalis (PPD). Antiserum against human LH stained PAS-positive cells located in the most ventral zone of the PPD and around the pars intermedia (PI). The distribution of the different cell types is similar to that of other teleosts. The phylogenetic implications of the degree of cross-reactivity of the antisera against human pituitary hormones with specific cells of the teleost fish pituitary is discussed.