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

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

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+

Anisakis Allergy: Is Aquacultured Fish a Safe and Alternative Food to Wild-Capture Fisheries for Anisakis simplex-Sensitized Patients?

Background: Anisakis simplex (A. simplex) infection, in humans, causes a series of clinical manifestations affecting the gastro-intestinal tract known as Anisakiasis/Anisakidosis. Patients may also present allergic manifestations such as hives and/or angioedema and even anaphylactic shock. The aim of this study was to investigate whether aquacultured fish could be considered A.simplex-free food and constitute a safe, alternative, wild-capture fish food for Gastro-Allergic Anisakiasis (GAA)-sensitized subjects. Methods: Protein extracts from A. simplex larvae in the third stage (L3) and from edible part of heavily infected horse mackerel (Trachurus trachurus) and aquacultured sea bream, have been tested for A. simplex allergens presence by immunological analysis. Western blot analysis using, as source of specific Anisakis allergens antibodies, serum samples from subjects referring allergic symptoms after raw fish ingestion, was performed. These subjects showed high levels of specific IgE anti A.simplex allergens determined by clinical laboratory tests (ISAC test). Results: Our data demonstrate the presence of Ani s4 allergen in both infected and aquacultured fish extracts, providing a possible interpretation for the allergic manifestations reported by subjects, already sensitized to A. simplex, who ate frozen or well-cooked or, even, aquacultured fish. Conclusions: The present data stimulate more accurate prophylaxis suggestions for Anisakis allergy and more specific controls of fishmeal used in aquaculture