Ca2+ Homeostasis in the Agonist-sensitive Internal Store: Functional Interactions Between Mitochondria and the ER Measured In Situ in Intact Cells

Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals

Termination of cAMP signals by Ca2+ and Gαi via extracellular Ca2+ sensors: a link to intracellular Ca2+ oscillations

Termination of cyclic adenosine monophosphate (cAMP) signaling via the extracellular Ca2+-sensing receptor (CaR) was visualized in single CaR-expressing human embryonic kidney (HEK) 293 cells using ratiometric fluorescence resonance energy transfer–dependent cAMP sensors based on protein kinase A and Epac. Stimulation of CaR rapidly reversed or prevented agonist-stimulated elevation of cAMP through a dual mechanism involving pertussis toxin–sensitive Gαi and the CaR-stimulated increase in intracellular [Ca2+]. In parallel measurements with fura-2, CaR activation elicited robust Ca2+ oscillations that increased in frequency in the presence of cAMP, eventually fusing into a sustained plateau. Considering the Ca2+ sensitivity of cAMP accumulation in these cells, lack of oscillations in [cAMP] during the initial phases of CaR stimulation was puzzling. Additional experiments showed that low-frequency, long-duration Ca2+ oscillations generated a dynamic staircase pattern in [cAMP], whereas higher frequency spiking had no effect. Our data suggest that the cAMP machinery in HEK cells acts as a low-pass filter disregarding the relatively rapid Ca2+ spiking stimulated by Ca2+-mobilizing agonists under physiological conditions

Extracellular calcium acts as a “third messenger” to regulate enzyme and alkaline secretion

It is generally assumed that the functional consequences of stimulation with Ca2+-mobilizing agonists are derived exclusively from the second messenger action of intracellular Ca2+, acting on targets inside the cells. However, during Ca2+ signaling events, Ca2+ moves in and out of the cell, causing changes not only in intracellular Ca2+, but also in local extracellular Ca2+. The fact that numerous cell types possess an extracellular Ca2+ “sensor” raises the question of whether these dynamic changes in external [Ca2+] may serve some sort of messenger function. We found that in intact gastric mucosa, the changes in extracellular [Ca2+] secondary to carbachol-induced increases in intracellular [Ca2+] were sufficient and necessary to elicit alkaline secretion and pepsinogen secretion, independent of intracellular [Ca2+] changes. These findings suggest that extracellular Ca2+ can act as a “third messenger” via Ca2+ sensor(s) to regulate specific subsets of tissue function previously assumed to be under the direct control of intracellular Ca2+

A Reassessment of the Effects of Luminal [Ca2+] on Inositol 1,4,5-Trisphosphate-induced Ca2+ Release from Internal Stores

Inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ release from intracellular stores displays complex kinetic behavior. While it well established that cytosolic [Ca2+] can modulate release by acting on the InsP3 receptor directly, the role of the filling state of internal Ca2+stores in modulating Ca2+ release remains unclear. Here we have reevaluated this topic using a technique that permits rapid and reversible changes in free [Ca2+] in internal stores of living intact cells without altering cytoplasmic [Ca2+], InsP3 receptors, or sarcoendoplasmic reticulum Ca2+ ATPases (SERCAs). N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), a membrane-permeant, low affinity Ca2+ chelator was used to manipulate [Ca2+] in intracellular stores, while [Ca2+] changes within the store were monitored directly with the low-affinity Ca2+ indicator, mag-fura-2, in intact BHK-21 cells. 200 microM TPEN caused a rapid drop in luminal free [Ca2+] and significantly reduced the extent of the response to stimulation with 100 nm bradykinin, a calcium-mobilizing agonist. The same effect was observed when intact cells were pretreated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid(acetoxymethyl ester) (BAPTA-AM) to buffer cytoplasmic [Ca2+] changes. Although inhibition of Ca2+ uptake using the SERCA inhibitor tBHQ permitted significantly larger release of Ca2+ from stores, TPEN still attenuated the release in the presence of tBHQ in BAPTA-AM-loaded cells. These results demonstrate that the filling state of stores modulates the magnitude of InsP3-induced Ca2+release by additional mechanism(s) that are independent of regulation by cytoplasmic [Ca2+] or effects on SERCA pumps

Introducing the Bulletin of Atmospheric Science and Technology

The rapid technological development of the past few decades has allowed for an unprecedented wealth of data about ourselves and our planet. The cost reduction of space platforms, the microelectronic revolution and the nearly exponential increase in computer power have been generating novel opportunities to explore and understand the world around us. Tools and theoretical approaches, capable of putting together all the insights we may possibly gain from all these new streams of data in a multidisciplinary framework, are still being developed. We are hence faced with both a unique challenge and an opportunity to make a significant progress in many scientific fields, first and foremost in the atmospheric and climate sciences. We are pleased to announce here the launch of the Bulletin of Atmospheric Science and Technology (BAST), a new peer-reviewed journal which is meant to bridge this gap in the broad area of the atmospheric sciences. The journal encourages a cross-disciplinary approach with an emphasis on new sensor technologies and systems, combined observational and modeling techniques, innovative numerical methods, data analysis, and retrieval techniques. BAST offers a platform to share new ideas and fresh developments to stimulate research activities focusing on urban, coastal, marine, rural, and mountain environments. Particular attention will be given to cross-disciplinary studies, especially those involving citizens for the collection of crowd-sourced data and those devoted to the characterization of uncertainties and homogenization of methods. BAST aims at connecting weather and climate communities using both observational and modeling approaches, creating a forum hosting discussion and brainstorming activities. The journal also hopes to attract contributions reporting approaches or techniques from other scientific fields that can be applicable to atmospheric sciences, as well as contributions where technological developments are discussed alongside with their scientific and societal impacts. In this sense BAST will provide a new platform to support the technological revolution towards a climate-smart society through the collection and exploitation of big data. The journal will give visibility to international experiments and projects in atmospheric science and technology, illustrating preliminary or consolidated results from these initiatives. Additional fields of interest are : environmental protection; observation, understanding, and modeling of hazardous and extreme events and mitigation of their impacts; development of new sensing tools integrating satellite information with surface or airborne measurements; operation of unmanned and remotely piloted air vehicles equipped with sensors of small size and weight, especially remote sensors, pushing electro-optical-mechanical components towards a continuously increasing miniaturization. Research articles, Review articles, Technical reports, Brief reports, Letters and News are welcome. While keeping the focus of the journal on scientific research, the “Bulletin” format provides appropriate visibility to contributions from the operational side, i.e., meteorological services and private companies developing sensors and products of interest to the atmospheric science and technology community. Below we provide a more detailed description of the topics that will be emphasized and fostered in BAST

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP). Methods/Principal Findings: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit $RI\beta$ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. Conclusions: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events

Kinetics of epsilon antitoxin antibodies in different strategies for active immunization of lambs against enterotoxaemia

Enterotoxaemia, a common disease that affects domestic small ruminants, is mainly caused by the epsilon toxin of Clostridium perfringens type D. The present study tested four distinct immunization protocols to evaluate humoral response in lambs, a progeny of non-vaccinated sheep during gestation. Twenty-four lambs were randomly allocated into four groups according to age (7, 15, 30 and 45 days), receiving the first dose of epsilon toxoid commercial vaccine against clostridiosis with booster after 30 days post vaccination. Indirect ELISA was performed after the first vaccine dose and booster to evaluate the immune response of the lambs. Results showed that for the four protocols tested all lambs presented serum title considered protective (≥0.2UI/ml epsilon antitoxin antibodies) and also showed that the anticipation of primovaccination of lambs against enterotoxaemia conferred serum title considered protective allowing the optimization of mass vaccination of lambs

Permeability pathways for non-electrolytes through Bufo bufo gall-bladder

Amphotericin B treatment increases the thiourea, D-xylose and mannitol fluxes and lowers those of urea, N-methyl-urea, acetamide, formamide, and N-N'-dimethyl-thiourea. The degree of flux inhibition is related to the cellular permeability of these compounds. Most probably Amphotericin B increases the permeability of all those molecules across the luminal plasma membrane, but simultaneously elicits a cellular swelling, which reduces the diffusion across the lateral plasma membranes. This effect masks the polyene effect especially for molecules showing a mainly cellular permeation pathway such as amides and lipid soluble molecules

Effect of cycloheximide on urea facilitated transport through toad gallbladder epithelium

Transepithelial urea outfluxes across toad gallbladder were determined before and after the addition of cycloheximide. The drug inhibits the movement of urea but has no effect on thiourea and antipyrine outfluxes. The inhibition of amide transport is time dependent as also shown in counterflow experiments. These results are consistent with the hypothesis that cycloheximide inhibits the synthesis of membrane proteic sites involved in urea mediated transport

Facilitated transport of urea across the gall-bladder luminal membrane

Counterflow experiments demonstrate the existence of urea counter-transport on the epithelium luminal surface. This phenomenon disappears when 10(-4) M phloretin is added to the perfusion fluid. Moreover counterflow experiments made using thiourea as elicitor, demonstrate that the phenomenon is specific for the urea