The effect of angiotensin II on intracellular pH is mediated by AT(1) receptor translocation

The effect of ANG II on intracellular pH (pH(i)) recovery rate and AT(1) receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT(1)) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 +/- 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 +/- 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10(-9) M) but not with ANG II (10(-6) M). Losartan (10(-7) M) and dimethyl-BAPTA-AM (10(-7) M) decreased significantly the stimulatory effect of ANG II (10(-9) M) and induced an increase in Na+/H+ exchanger 1 (NHE-1) activity with ANG II (10(-6) M). Immunofluorescence studies indicated that in control situation, the hAT(1) receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10(-9) M) and internalized with ANG II (10(-6) M). Losartan (10(-7) M) induced hAT(1) translocation to plasma membrane in all studied groups. Dimethyl-BAPTA-AM (10(-7) M) did not change the effect of ANG II (10(-9) M) on the hAT(1) receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10(-6) M). With ionomycin (10(-6) M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT(1) receptor and intracellular signaling events related to AT(1) translocation.

Lack of photoreceptor signaling alters the expression of specific synaptic proteins in the retina

Synaptic modulation by activity-dependent changes constitutes a cellular mechanism for neuronal plasticity. However, it is not clear how the complete lack of neuronal signaling specifically affects elements involved in the communication between neurons. In the retina, it is now well established that both chemical and electrical synapses are essential to mediate the transmission of visual signaling triggered by the photoreceptors. In this study, we compared the expression of synaptic proteins in the retinas of wild-type (WT) vs. rd/rd mice, an animal model that displays inherited and specific ablation of photoreceptors caused by a mutation in the gene encoding the beta-subunit of rod cGMP-phosphodiesterase (Pde6b(rd1)). We specifically examined the expression of connexins (Cx), the proteins that form the gap junction channels of electrical synapses, in addition to synaptophysin and synapsin 1, which are involved in the release of neurotransmitters at chemical synapses. Our results revealed that Cx36 gene expression levels are lower in the retinas of rd/rd when compared with WT. Confocal analysis indicated that Cx36 immunolabeling almost disappeared in the outer plexiform layer without significant changes in protein distribution within the inner plexiform layer of rd/rd retinas. Likewise, synaptophysin expression remarkably decreased in the outer plexiform layer of rd/rd retinas, and this down-regulation was also associated with diminished transcript levels. Furthermore, we observed down-regulation of Cx57 gene expression in rd/rd retinas when compared with WT and also changes in protein distribution. Interestingly, Cx45 and synapsin I expression in rd/rd retinas showed no noticeable changes when compared with WT. Taken together, our results revealed that the loss of photoreceptors leads to decreased expression of some synaptic proteins. More importantly, this study provides evidence that neuronal activity regulates, but is not essential to maintain, the expression of synaptic elements. (c) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.

Cell death and lumen formation in spheroids of MCF-7 cells

3D (three-dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F-actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF-7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Three-dimensional Reconstruction of Ovaries of Leaf-cutting Ant (Atta sexdens rubropilosa) Queens (Hymenoptera: Formicidae)

In this study, the ovary morphology of newly emerged ant queens of Atta sexdens rubropilosa was studied in whole mount preparations by confocal microscopy. The ovaries are composed of approximately 40 ovarioles, showing non-synchronic oocyte maturation. The terminal filament with clusters of undifferentiated cells was found at the distal end of the ovarioles. Next to this region is the germarium, composed of several elongated cystocytes interconnected by cytoplasmic bridges. The nurse cells (23-28 cells) result from asymmetric mitosis. Cytoskeleton analysis showed F-actin concentrated at the muscle cells of the external tunica and in fusomes inside the ovarioles. Microtubules were concentrated around the nuclei of the nurse and follicular cells. In contrast, the oocytes and the external tunica showed faint staining for tubulin.

Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography

Objectives: The aim of this study was to evaluate the genotoxic effects of X-rays on epithelial gingival cells during panoramic dental radiography using a differentiated protocol for the micronucleus test. Methods: 40 healthy individuals who underwent this procedure for diagnostic purposes on request from their dentists agreed to participate in this study. All of them answered a questionnaire before the examination. Epithelial gingival cells were obtained from the keratinized mucosa of the upper dental arcade by gentle scraping with a cervical brush immediately before exposure and 10 days later. Cytological preparations were stained according to the Feulgen-Rossenbeck reaction, counterstained with fast green 1% for 1 min and analysed under a light microscope. Micronuclei, nuclear projections (broken eggs) and degenerative nuclear alterations (pyknosis, karyolysis, karyorrhexis and condensed chromatin) were scored. Results: The frequency of micronuclei was significantly higher after exposure (P < 0.05), as were frequencies of nuclear alterations indicate of apoptosis (P < 0.001). Conclusions: These results indicate that X-ray radiation emitted during panoramic dental radiography induces a genotoxic effect on epithelial gingival cells that increases the frequency of chromosomal damage and nuclear alterations indicative of apoptosis.