A ação aguda direta da Ang-(1-7) sobre o fluxo reabsortivo de bicarbonato (JHCO3-) foi avaliada por microperfusão estacionária in vivo em túbulos proximais de ratos utilizando microelétrodo sensível a H+. Em ratos WKY, a Ang-(1-7) tem efeito bifásico sobre o NHE3: em baixas concentrações causa queda do JHCO3-, enquanto que em altas concentrações estimula o JHCO3-. Em ratos SHR, a Ang-(1-7) demonstrou efeitos opostos aos dos ratos WKY; a Ang-(1-7; 10-9 M) aumentou o JHCO3-, enquanto a Ang (1-7; 10-6 M) causou significante queda do JHCO3-. Em adição, monitoramos fluorimétricamente a concentração de cálcio citosólico ([Ca2+]i) em túbulos proximais isolados, por meio do probe sensivél a cálcio FURA-2-AM. Nossos dados indicam que a [Ca2+]i em ratos WKY no controle é 99,7 ± 2,28 nM; a Ang-(1-7) aumenta esse valor. Em animais SHR o controle é 94,3 ± 1,66 nM; a Ang-(1-7) aumenta esse valor. Em conclusão, nossos dados indicam que a interação dos efeitos opostos dose dependente da Ang-(1-7) e Ang II sobre JHCO3- e a [Ca2+]i representa importante mecanismo de regulação fisiológica do volume e do pH intra-extracelular. / Direct acute effects of Ang-(1-7) on the resorptive bicarbonate flow (JHCO3-) was evaluated by stationary microperfusion in vivo in proximal tubules using microelectrode sensitive to H+. In WKY control rats, Ang-(1-7) has biphasic effects on Na+/H+ exchanger isoform 3 (NHE3): at low dose inhibits and at higher one stimulates the NHE3. In SHR rats, Ang-(1-7) displayed opposite effects on SHR when compared to WKY rats: Ang- (1-7) at low dose increases JHCO3- whereas Ang (1-7) at higher dose reduces JHCO3-. In addition, we have fluorimetrically monitored cytosolic calcium ([Ca2+]i) in isolated proximal tubule through FURA-2-AM. Our data show that [Ca2+]i in the WKY control group is 99.7 ± 2.28 nM. Ang-(1-7) at 10-9 or 10-6 M increases [Ca2+]i. In SHR control rats [Ca2+]i is 94.3 ± 1.66 nM; Ang-(1-7) increase [Ca2+]i. Thus, our data are indicating that the interaction of the opposing dose-dependent effects of Ang-(1-7) and Ang II on JHCO3- and [Ca2+]i may represent an important physiological mechanism for regulating the intra and extracellular volume and pH in normotensive and hypertensive individuals.

Electrical potentials exist across the membranes of nearly every cell type in the body. In addition, excitable cells, such as neurons, myocytes and even some endocrine cells elicit electrochemical fluctuations, action potentials (AP), in the cell membrane to initiate cell-to-cell communication or intracellular processes. The basis for the electrical potential is rooted within an array of complex interactions between monovalent ions and their associated membrane channels and transporters that regulate the flux of these charged species across the hydrophobic bilayer. Here, an expansion of our recently published work [1] will serve to explore the modern concepts regarding the origin of the AP as well as to examine the mechanisms by which intracellular calcium ([Ca2+]i) is regulated within the HL-1 mouse cardiac myocyte.

no / Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μM LPA in 60 μM Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mM Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway.

AMP-activated protein kinase (AMPK) is known as a pivotal cellular energy sensor, mediating the adaptation to low energy levels by deactivating anabolic processes and activating catabolic processes in order to restore the cellular ATP supply when the cellular AMP/ATP ratio is increased. Besides this well-known role, it has also been shown to exert protective effects under hypoxia. While an insufficient supply with oxygen might easily deplete cellular energy levels, i.e., ATP concentration, manifold other mechanisms have been suggested and are heavily disputed regarding the activation of AMPK under hypoxia independently from cellular AMP concentrations. However, an activation of AMPK preceding energy depletion could induce a timely adaptation reaction preventing more serious damage. A connection between AMPK and the master regulator of hypoxic adaptation via gene transcription, hypoxia-inducible factor (HIF), has also been taken into account, orchestrating their concerted protective action. This review will summarize the current knowledge on mechanisms of AMPK activation under hypoxia and its interrelationship with HIF.