In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

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Biological properties of extracellular vesicles and their physiological functions

The recently discovered epithelial sodium channel (ENaC)/degenerin (DEG) gene family encodes sodium channels involved in various cell functions in metazoans. Subfamilies found in invertebrates or mammals are functionally distinct. The degenerins in Caenorhabditis elegans participate in mechanotransduction in neuronal cells, FaNaC in snails is a ligand-gated channel activated by neuropeptides, and the Drosophila subfamily is expressed in gonads and neurons. In mammals, ENaC mediates Na+ transport in epithelia and is essential for sodium homeostasis. The ASIC genes encode proton-gated cation channels in both the central and peripheral nervous system that could be involved in pain transduction. This review summarizes the physiological roles of the different channels belonging to this family, their biophysical and pharmacological characteristics, and the emerging knowledge of their molecular structure. Although functionally different, the ENaC/DEG family members share functional domains that are involved in the control of channel activity and in the formation of the pore. The functional heterogeneity among the members of the ENaC/DEG channel family provides a unique opportunity to address the molecular basis of basic channel functions such as activation by ligands, mechanotransduction, ionic selectivity, or block by pharmacological ligands.

https://www.physiology.org/doi/pdf/10.1152/physrev.00007.2002

Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene result in defective epithelial cAMP-dependent Cl− secretion and increased airway Na+ absorption. The mechanistic links between these altered ion transport processes and the pathogenesis of cystic fibrosis lung disease, however, are unclear. To test the hypothesis that accelerated Na+ transport alone can produce cystic fibrosis-like lung disease, we generated mice with airway-specific overexpression of epithelial Na+ channels (ENaC). Here we show that increased airway Na+ absorption in vivo caused airway surface liquid (ASL) volume depletion, increased mucus concentration, delayed mucus transport and mucus adhesion to airway surfaces. Defective mucus transport caused a severe spontaneous lung disease sharing features with cystic fibrosis, including mucus obstruction, goblet cell metaplasia, neutrophilic inflammation and poor bacterial clearance. We conclude that increasing airway Na+ absorption initiates cystic fibrosis-like lung disease and produces a model for the study of the pathogenesis and therapy of this disease.

Increased airway epithelial Na+ absorption produces cystic fibrosis-like lung disease in mice.

Electroneutral cation-Cl− cotransporters compose a family of solute carriers in which cation (Na+ or K+) movement through the plasma membrane is always accompanied by Cl− in a 1:1 stoichiometry. Se...

https://www.physiology.org/doi/pdf/10.1152/physrev.00011.2004

Molecular Physiology and Pathophysiology of Electroneutral Cation-Chloride Cotransporters

Aldosterone stimulates sodium transport in the renal collecting duct by activating the epithelial sodium channel (ENaC). To investigate the basis of this effect, we have developed a novel set of rabbit polyclonal antibodies to the 3 subunits of ENaC and have determined the abundance and distribution of ENaC subunits in the principal cells of the rat renal collecting duct. Elevated circulating aldosterone (due to either dietary NaCl restriction or aldosterone infusion) markedly increased the abundance of alphaENaC protein without increasing the abundance of the beta and gamma subunits. Thus, alphaENaC is selectively induced by aldosterone. In addition, immunofluorescence immunolocalization showed a striking redistribution in ENaC labeling to the apical region of the collecting duct principal cells. Finally, aldosterone induced a shift in molecular weight of gammaENaC from 85 kDa to 70 kDa, consistent with physiological proteolytic clipping of the extracellular loop as postulated previously. Thus, at the protein level, the response of ENaC to aldosterone stimulation is heterogenous, with both quantitative and qualitative changes that can explain observed increases in ENaC-mediated sodium transport.

/pdf/aldosterone-mediated-regulation-of-enac-a-b-and-g-subunit-3rjegubcdo.pdf

Aldosterone-mediated regulation of ENaC α, β, and γ subunit proteins in rat kidney

Although the collecting duct is regarded as the primary site at which mineralocorticoids regulate renal sodium transport in the kidney, recent evidence points to the distal convoluted tubule as a possible site of mineralocorticoid action. To investigate whether mineralocorticoids regulate the expression of the thiazide-sensitive Na–Cl cotransporter (TSC), the chief apical sodium entry pathway of distal convoluted tubule cells, we prepared an affinity-purified, peptide-directed antibody to TSC. On immunoblots, the antibody recognized a prominent 165-kDa band in membrane fractions from the renal cortex but not from the renal medulla. Immunofluorescence immunocytochemistry showed TSC labeling only in distal convoluted tubule cells. Semiquantitative immunoblotting studies demonstrated a large increase in TSC expression in the renal cortex of rats on a low-NaCl diet (207 ± 21% of control diet). Immunofluorescence localization in tissue sections confirmed the strong increase in TSC expression. Treatment of rats for 10 days with a continuous subcutaneous infusion of aldosterone also increased TSC expression (380 ± 58% of controls). Furthermore, 7-day treatment of rats with an orally administered mineralocorticoid, fludrocortisone, increased TSC expression (656 ± 114% of controls). We conclude that the distal convoluted tubule is an important site of action of the mineralocorticoid aldosterone, which strongly up-regulates the expression of TSC.

https://www.pnas.org/content/pnas/95/24/14552.full.pdf

The thiazide-sensitive Na–Cl cotransporter is an aldosterone-induced protein

Sodium transport is increased by vasopressin in the cortical collecting ducts of rats and rabbits. Here we investigate, by quantitative immunoblotting, the effects of vasopressin on abundances of the epithelial sodium channel (ENaC) subunits (alpha, beta, and gamma) in rat kidney. Seven-day infusion of 1-deamino-[8-D-arginine]-vasopressin (dDAVP) to Brattleboro rats markedly increased whole kidney abundances of beta- and gamma-ENaC (to 238% and 288% of vehicle, respectively), whereas alpha-ENaC was more modestly, yet significantly, increased (to 142% of vehicle). Similarly, 7-day water restriction in Sprague-Dawley rats resulted in significantly increased abundances of beta- and gamma- but no significant change in alpha-ENaC. Acute administration of dDAVP (2 nmol) to Brattleboro rats resulted in modest, but significant, increases in abundance for all ENaC subunits, within 1 h. In conclusion, all three subunits of ENaC are upregulated by vasopressin with temporal and regional differences. These changes are too slow to play a major role in the short-term action of vasopressin to stimulate sodium reabsorption in the collecting duct. Long-term increases in ENaC abundance should add to the short-term regulatory mechanisms (undefined in this study) to enhance sodium transport in the renal collecting duct.

Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney

Epithelial sodium channel (ENaC) subunit (α, β, and γ) mRNA and protein have been localized to the principal cells of the connecting tubule (CNT), cortical collecting duct (CCD), and outer medullar...

https://www.physiology.org/doi/pdf/10.1152/ajprenal.2001.280.6.F1093

Immunocytochemical and immunoelectron microscopic localization of α-, β-, and γ-ENaC in rat kidney

We carried out semiquantitative immunoblotting of kidney to identify apical sodium transporter proteins whose abundances are regulated by angiotensin II. In NaCl-restricted rats (0.5 mEq Na/200 g BW/d), the type 1 angiotensin II receptor (AT1 receptor) antagonist, candesartan, (1 mg/kg of body weight per day SC for 2 days) markedly decreased the abundance of the subunit of the epithelial sodium channel (ENaC). This subunit has been shown to be rate-limiting for assembly of mature ENaC complexes. In addition, systemic infusion of angiotensin II increased ENaC protein abundance in rat kidney cortex. The decrease in ENaC protein abundance in response to AT1 receptor blockade was associated with a fall in ENaC mRNA abundance (real-time RT-PCR), consistent with transcriptionally mediated regulation. The effect of AT1 receptor blockade on ENaC expression was not blocked by spironolactone, suggesting a direct role of the AT1 receptor in regulation of ENaC gene expression. Candesartan administration was also found to increase the abundances of the and subunits. The increase in and ENaC protein abundance was not associated with a significant increase in the renal abundances of the corresponding mRNAs, suggesting a posttranscriptional mechanism. Immunocytochemistry confirmed the increase in and ENaC protein abundance and demonstrated candesartan-induced ENaC internalization in collecting duct cells. The results support the view that the angiotensin II receptor regulates ENaC abundance, consistent with a role for angiotensin II in regulation of collecting duct function. (Hypertension. 2003;41:1143-1150.)

Shyama Masilamani

Papers

Aldosterone-mediated regulation of ENaC α, β, and γ subunit proteins in rat kidney

The thiazide-sensitive Na–Cl cotransporter is an aldosterone-induced protein

Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney

Immunocytochemical and immunoelectron microscopic localization of α-, β-, and γ-ENaC in rat kidney

Long-Term Regulation of ENaC Expression in Kidney by Angiotensin II