Abstract
BackgroundReactive oxygen (ROS) and nitrogen (RNS) species are produced during normal unstressed metabolic activity in aerobic tissues. Most analytical work uses tissue homogenates, and lacks spatial information on the tissue specific sites of actual ROS formation. Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes at cellular level.ResultsTogether with oxidative stress measurements, here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of ROS and RNS formation in the living tissue. Our results indicate that a) mitochondria located in the basal parts of the epithelial cells close to the blood vessels are hyperpolarized with high Δψm, whereas b) the peripheral mitochondria close to the cilia have low (depolarized) Δψm. These mitochondria are densely packed (mitotracker Deep Red 633 staining), have acidic pH (Ageladine-A) and collocate with high formation of nitric oxide (DAF-2DA staining). NO formation is also observed in the endothelial cells surrounding the filament blood sinus. ROS (namely H2O2, HOO• and ONOO− radicals, assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. On the ventral bend of the gills, subepithelial mucus glands contain large mucous vacuoles showing higher fluorescence intensities for O2•- than the rest of the tissue. Whether this O2•- production is instrumental to mucus formation or serves antimicrobial protection of the gill surface is unknown. Cells of the ventral bends contain the superoxide forming mucocytes and show significantly higher protein carbonyl formation than the rest of the gill tissue.ConclusionsIn summary, ROS and RNS formation is highly compartmentalized in bivalve gills under unstressed conditions. The main mechanisms are the differentiation of mitochondria membrane potential and basal ROS formation in inner and outer filament layers, as well as potentially antimicrobial ROS formation in the central blood vessel. Our results provide new insight into this subject and highlight the fact that studying ROS formation in tissue homogenates may not be adequate to understand the underlying mechanism in complex tissues.
Highlights
Reactive oxygen (ROS) and nitrogen (RNS) species are produced during normal unstressed metabolic activity in aerobic tissues
Under unstressed conditions Reactive oxygen and nitrogen species (ROS) and RNS are produced in very low quantities, and bulk formation of ROS is usually measured in mitochondrial isolates [7, 8], cell preparations [9, 10], or even in tissue homogenates (e.g. [11, 12])
To our knowledge this study provides the first record of the mitochondrial spatial distribution and the basal production patterns of different ROS/RNS in bivalve gill tissues
Summary
Reactive oxygen (ROS) and nitrogen (RNS) species are produced during normal unstressed metabolic activity in aerobic tissues. In living tissues steady state ROS concentrations are highly compartmentalized, and the distribution patterns of different ROS and RNS across subcellular structures can be the clue to their biochemical role and function. This compartmentalization can be visualized using ROS/RNS sensitive probes, molecules which emit fluorescence upon reaction with the active species and after excitation at a distinct wavelength. Fluorescent probes enable minimal invasive detection of very small ROS/RNS quantities in living cells and tissues and facilitate the understanding of the dynamics and function of ROS production. This type of studies may help by providing input on the amount of ROS/RNS (in a qualitative manner) and setting the bases for further studies aiming to distinguish signaling or biosynthetic functions from toxic overproduction of ROS/RNS during an acute stress response
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