The Role of Cytokines in the Functional Activity of Phagocytes in Blood and Colostrum of Diabetic Mothers

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Production and characterization of natural rubber-Ca/P blends for biomedical purposes

Conselho Nacional de Desenvolvimento Cient��fico e Tecnol��gico (CNPq)

L-Aminoacid oxidase from bothrops leucurus venom Induces nephrotoxicity via apoptosis and necrosis

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

STIM 1/Orai 1 contributes to sex differences in vascular responses to calcium in spontaneously hypertensive rats

Sex differences in Ca2+-dependent signalling and homoeostasis in the vasculature of hypertensive rats are well characterized. However, sex-related differences in SOCE (store-operated Ca2+ entry) have been minimally investigated. We hypothesized that vascular protection in females, compared with males, reflects decreased Ca2+ mobilization due to diminished activation of Orai 1/STIM 1 (stromal interaction molecule I). In addition, we investigated whether ovariectomy in females affects the activation of the Orai 1/STIM 1 pathway. Endothelium-denuded aortic rings from male and female SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar Kyoto) rats and from OVX (ovariectomized) or sham female SHRSP and WKY rats were used to functionally evaluate Ca2+ influx-induced contractions. Compared with females, aorta from male SHRSP displayed: (i) increased contraction during the Ca2+-loading period; (ii) similar transient contraction during Ca2+ release from the intracellular stores; (iii) increased activation of STIM 1 and Orai1, as shown by the blockade of STIM 1 and Orai1 with neutralizing antibodies, which reversed the sex differences in contraction during the Ca2+-loading period; and (iv) increased expression of STIM I and Orai I. Additionally, we found that aortas from OVX-SHRSP showed increased contraction during the Ca2+-loading period and increased Orai1 expression, but no changes in the SR (sarcoplasmic reticulum)-buffering capacity or STIM I expression. These findings suggest that augmented activation of STIM 1/Orai 1 in aortas from male SHRSP represents a mechanism that contributes to sex-related impaired control of intracellular Ca2+ levels. Furthermore, female sex hormones may negatively modulate the STIM/Orai 1 pathway, contributing to vascular protection observed in female rats.

Interlinked dual-time feedback loops can enhance robustness to stochasticity and persistence of memory.

Multiple interlinked positive feedback loops shape the stimulus responses of various biochemical systems, such as the cell cycle or intracellular Ca2+ release. Recent studies with simplified models have identified two advantages of coupling fast and slow feedback loops. This dual-time structure enables a fast response while enhancing resistances of responses and bistability to stimulus noise. We now find that (1) the dual-time structure similarly confers resistance to internal noise due to molecule number fluctuations, and (2) model variants with altered coupling, which better represent some specific biochemical systems, share all the above advantages. We also develop a similar bistable model with coupling of a fast autoactivation loop to a slow loop. This model's topology was suggested by positive feedback proposed to play a role in long-term synaptic potentiation (LTP). The advantages of fast response and noise resistance are also present in this autoactivation model. Empirically, LTP develops resistance to reversal over approximately 1h . The model suggests this resistance may result from increased amounts of synaptic kinases involved in positive feedback.

Rapid effects of 1alpha,25-dihydroxyvitamin D3 on osteoblasts: Studies of membrane transducers that affect calcium signals

1,25-dihydroxyvitamin D3 [1,25(OH)2D 3] exerts pleiotropic effects on osteoblasts via both long-term nuclear receptor-mediated and rapid membrane-initiated pathways during bone remodeling and mineral homeostasis. This study explored the membrane transducers that mediate rapid effects of 1,25(OH)2D3 on osteoblasts, including sphingomyelinase (SMase) and L-type voltage sensitive calcium channels (VSCCs). ^ It was previously demonstrated that 1,25(OH)2D3 stimulates transmembrane influx of Ca2+ through VSCCs in ROS 17/2.8 osteoblasts, however the molecular identity of 1,25(OH)2D 3-regulated VSCC has not been known. In this study, on the basis of in vitro tests of three unique ribozymes specifically cleaving a1C mRNA, I transfected ROS 17/2.8 cells with vectors coding recombinant ribozyme modified with U1 snRNA structure, and successfully selected stable clonal cells in which the expression of a1C was strikingly reduced. Ca2+ influx studies in these cells compared to control transfectants showed selective attenuation of depolarization- and 1,25(OH)2D3-regulated Ca2+ responses. These results allow us to conclude that the cardiac ( a1C ) subtype of the L-type VSCC is the major membrane transducer of Ca 2+ influx in osteoblasts. ^ I also demonstrated that 1,25(OH)2D3 induces a rapid hydrolysis of membrane sphingomyelin (SM) in ROS 17/2.8 cells, with the concomitant generation of ceramide, detectable at 15 minute, and maximal at 1 hour after addition. Sphingosine, sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC), downstream products of SM hydrolysis, but not ceramide, elicit Ca 2+ release from intracellular stores. Considering ceramide, sphingosine, and SPP as second messengers modulating intracellular kinases or phosphatases, these findings implicate sphingolipid-signaling pathways in transducing rapid effects of 1,25(OH)2D3 on osteoblasts. In structure/function analyses of sphingolipid signaling, it was observed that psychosine elicits Ca2+ release from intracellular stores. This challenges the dogma that sphingosine phosphorylation permits mobilization of Ca2+ , because psychosine is a sphingosine analog galactosylated at 1-carbon, preventing phosphorylation at that site. Psychosine is the pathological metabolite found in patients with Krabbe's disease, suggesting that psychosine disrupts the physiological sphingolipid signaling by chronic release of Ca2+ from intracellular stores. ^ Slower SM turnover than Ca2+ influx through VSCCs in response to 1,25(OH)2D3 demonstrates ceramide does not mediate the 1,25(OH)2D3-induced Ca2+ signaling, a conclusion endorsed further by the failure of ceramide to induce Ca 2+ signaling. ^

Development and characterization of a scaffold-free 3D spheroid model of iPSC-derived human cardiomyocytes

Purpose: Cardiomyocytes are terminally differentiated cells in the adult heart and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSC) from human origin was developed and characterized. Methods: Human cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs) were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. 2D cultures were examined using immunofluorescence microscopy and Western blotting while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. Results: iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to pro-hypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived cardiomyocytes formed spheroidal MTs within 4 days showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature, and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium-transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca2+-release, extracellular calcium levels. Conclusions: 3D culture using iPSC-derived human cardiomyocytes provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

Activation of nicotinic receptor-induced postsynaptic responses to luteinizing hormone-releasing hormone in bullfrog sympathetic ganglia via a Na+-dependent mechanism

Nicotine at very low doses (5���30 nM) induced large amounts of luteinizing hormone-releasing hormone (LHRH) release, which was monitored as slow membrane depolarizations in the ganglionic neurons of bullfrog sympathetic ganglia. A nicotinic antagonist, d-tubocurarine chloride, completely and reversibly blocked the nicotine-induced LHRH release, but it did not block the nerve-firing-evoked LHRH release. Thus, nicotine activated nicotinic acetylcholine receptors and produced LHRH release via a mechanism that is different from the mechanism for evoked release. Moreover, this release was not caused by Ca2+ influx through either the nicotinic receptors or the voltage-gated Ca2+ channels because the release was increased moderately when the extracellular solution was changed into a Ca2+-free solution that also contained Mg2+ (4 mM) and Cd2+ (200 ��M). The release did not depend on Ca2+ release from the intraterminal Ca2+ stores either because fura-2 fluorimetry showed extremely low Ca2+ elevation (���30 nM) in response to nicotine (30 nM). Moreover, nicotine evoked LHRH release when [Ca2+] elevation in the terminals was prevented by loading the terminals with 1,2-bis(2-aminophenoxy)ethane-N,N,N���,N���-tetraacetic acid and fura-2. Instead, the nicotine-induced release required extracellular Na+ because substitution of extracellular NaCl with N-methyl-d-glucamine chloride completely blocked the release. The Na+-dependent mechanism was not via Na+ influx through the voltage-gated Na+ channels because the release was not affected by tetrodotoxin (1���50 ��M) plus Cd2+ (200 ��M). Thus, nicotine at very low concentrations induced LHRH release via a Na+-dependent, Ca2+-independent mechanism.

Platelet signal transduction defect with G�� subunit dysfunction and diminished G��q in a patient with abnormal platelet���responses

G proteins play a major role in signal transduction upon platelet activation. We have previously reported a patient with impaired agonist-induced aggregation, secretion, arachidonate release, and Ca2+ mobilization. Present studies demonstrated that platelet phospholipase A2 (cytosolic and membrane) activity in the patient was normal. Receptor-mediated activation of glycoprotein (GP) IIb-IIIa complex measured by flow cytometry using antibody PAC-1 was diminished despite normal amounts of GPIIb-IIIa on platelets. Ca2+ release induced by guanosine 5���-[��-thio]triphosphate (GTP[��S]) was diminished in the patient���s platelets, suggesting a defect distal to agonist receptors. GTPase activity (a function of ��-subunit) in platelet membranes was normal in resting state but was diminished compared with normal subjects on stimulation with thrombin, platelet-activating factor, or the thromboxane A2 analog U46619. Binding of 35S-labeled GTP[��S] to platelet membranes was decreased under both basal and thrombin-stimulated states. Iloprost (a stable prostaglandin I2 analog) -induced rise in cAMP (mediated by G��s) and its inhibition (mediated by G��i) by thrombin in the patient���s platelet membranes were normal. Immunoblot analysis of G�� subunits in the patient���s platelet membranes showed a decrease in G��q (<50%) but not G��i, G��z, G��12, and G��13. These studies provide evidence for a hitherto undescribed defect in human platelet G-protein ��-subunit function leading to impaired platelet responses, and they provide further evidence for a major role of G��q in thrombin-induced responses.

From calcium blips to calcium puffs: Theoretical analysis of the requirements for interchannel communication

In the cytoplasm of cells of different types, discrete clusters of inositol 1,4,5-trisphosphate-sensitive Ca2+ channels generate Ca2+ signals of graded size, ranging from blips, which involve the opening of only one channel, to moderately larger puffs, which result from the concerted opening of a few channels in the same cluster. These channel clusters are of unknown size or geometrical characteristics. The aim of this study was to estimate the number of channels and the interchannel distance within such a cluster. Because these characteristics are not attainable experimentally, we performed computer stochastic simulations of Ca2+ release events. We conclude that, to ensure efficient interchannel communication, as experimentally observed, a typical cluster should contain two or three tens of inositol 1,4,5-trisphosphate-sensitive Ca2+ channels in close contact.

Elemental propagation of calcium signals in response-specific patterns determined by environmental stimulus strength

Plant cells can respond qualitatively and quantitatively to a wide range of environmental signals. Ca2+ is used as an intracellular signal for volume regulation in response to external osmotic changes. We show here that the spatiotemporal patterns of hypo-osmotically induced Ca2+ signals vary dramatically with stimulus strength in embryonic cells of the marine alga Fucus. Biphasic or multiphasic Ca2+ signals reflect Ca2+ elevations in distinct cellular domains. These propagate via elemental Ca2+ release in nuclear or peripheral regions that are rich in endoplasmic reticulum. Cell volume regulation specifically requires Ca2+ elevation in apical peripheral regions, whereas an altered cell division rate occurs only in response to stimuli that cause Ca2+ elevation in nuclear regions.

Fire���diffuse���fire model of dynamics of intracellular calcium waves

When Ca2+ is released from internal stores in living cells, the resulting wave of increased concentration can travel without deformation (continuous propagation) or with burst-like behavior (saltatory propagation). We analyze the ���fire���diffuse���fire��� model in order to illuminate the differences between these two modes of propagation. We show that the Ca2+ release wave in immature Xenopus oocytes and cardiac myocytes is saltatory, whereas the fertilization wave in the mature oocyte is continuous.

Mitochondrial control of calcium-channel gating: A mechanism for sustained signaling and transcriptional activation in T lymphocytes

In addition to their well-known functions in cellular energy transduction, mitochondria play an important role in modulating the amplitude and time course of intracellular Ca2+ signals. In many cells, mitochondria act as Ca2+ buffers by taking up and releasing Ca2+, but this simple buffering action by itself often cannot explain the organelle's effects on Ca2+ signaling dynamics. Here we describe the functional interaction of mitochondria with store-operated Ca2+ channels in T lymphocytes as a mechanism of mitochondrial Ca2+ signaling. In Jurkat T cells with functional mitochondria, prolonged depletion of Ca2+ stores causes sustained activation of the store-operated Ca2+ current, ICRAC (CRAC, Ca2+ release-activated Ca2+). Inhibition of mitochondrial Ca2+ uptake by compounds that dissipate the intramitochondrial potential unmasks Ca2+-dependent inactivation of ICRAC. Thus, functional mitochondria are required to maintain CRAC-channel activity, most likely by preventing local Ca2+ accumulation near sites that govern channel inactivation. In cells stimulated through the T-cell antigen receptor, acute blockade of mitochondrial Ca2+ uptake inhibits the nuclear translocation of the transcription factor NFAT in parallel with CRAC channel activity and [Ca2+]i elevation, indicating a functional link between mitochondrial regulation of ICRAC and T-cell activation. These results demonstrate a role for mitochondria in controlling Ca2+ channel activity and signal transmission from the plasma membrane to the nucleus.

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca2+ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca2+ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca2+ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca2+ from ER, giving rise to a slow and small increase in [Ca2+]i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca2+ release by 4-CEP, giving rise to Ca2+ spikes. In glucose-stimulated beta cells forskolin induced Ca2+ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting ��TC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY2). We conclude that in situ activation of RY2 in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca2+ release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200���500 ��M ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, Po < 0.005) and brief (<250 ��s) gating events and insensitivity to Ca2+. Addition of ryanodine restores Ca2+-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.