A FRET-Based Approach for Quantitative Evaluation of Forskolin-Induced Pendrin Trafficking at the Plasma Membrane in Bronchial NCI H292 Cells

Tamma, Grazia; Ranieri, Marianna; Dossena, Silvia; Mise, Annarita Di; Nofziger, Charity; Svelto, María; Paulmichl, Markus; Valenti, Giovanna

doi:10.1159/000356639

Cited by 8 publications

(5 citation statements)

References 43 publications

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“…Previous studies have shown that tolvaptan impairs the increase in cAMP levels upon vasopressin action . Here, the effect of tolvaptan on cAMP level was evaluated with FRET‐based EPAC biosensors as already described . Specifically, the FRET probe contains cAMP‐binding sequence of Epac1 sandwiched between ECFP (donor) and EYFP (acceptor).…”

Section: Resultsmentioning

confidence: 99%

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment

Tamma

Mise

Ranieri

et al. 2017

J Cellular Molecular Medi

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Tolvaptan, a selective vasopressin V2 receptor antagonist, is a new generation diuretic. Its clinical efficacy is in principle due to impaired vasopressin‐regulated water reabsorption via aquaporin‐2 (AQP2). Nevertheless, no direct in vitro evidence that tolvaptan prevents AQP2‐mediated water transport, nor that this pathway is targeted in vivo in patients with syndrome of inappropriate antidiuresis (SIAD) has been provided. The effects of tolvaptan on the vasopressin–cAMP/PKA signalling cascade were investigated in MDCK cells expressing endogenous V2R and in mouse kidney. In MDCK, tolvaptan prevented dDAVP‐induced increase in ser256‐AQP2 and osmotic water permeability. A similar effect on ser256‐AQP2 was found in V1aR −/− mice, thus confirming the V2R selectively. Of note, calcium calibration in MDCK showed that tolvaptan per se caused calcium mobilization from the endoplasmic reticulum resulting in a significant increase in basal intracellular calcium. This effect was only observed in cells expressing the V2R, indicating that it requires the tolvaptan–V2R interaction. Consistent with this finding, tolvaptan partially reduced the increase in ser256‐AQP2 and the water permeability in response to forskolin, a direct activator of adenylyl cyclase (AC), suggesting that the increase in intracellular calcium is associated with an inhibition of the calcium‐inhibitable AC type VI. Furthermore, tolvaptan treatment reduced AQP2 excretion in two SIAD patients and normalized plasma sodium concentration. These data represent the first detailed demonstration of the central role of AQP2 blockade in the aquaretic effect of tolvaptan and underscore a novel effect in raising intracellular calcium that can be of significant clinical relevance.

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Section: Resultsmentioning

confidence: 99%

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment

Tamma

Mise

Ranieri

et al. 2017

J Cellular Molecular Medi

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“…CFP and YFP were excited at 436 and 500 nm, respectively; fluorescence emitted was measured at 480/40 nm for CFP and 535/30 nm for YFP and FRET. Corrected normalized FRET values were determined as already described (Rodighiero et al, 2008 ; Tamma et al, 2013 , 2017 ; Russo et al, 2017 ). Each image was corrected for CFP cross-talk and YFP cross-excitation.…”

Section: Methodsmentioning

confidence: 99%

Activation of the Calcium-Sensing Receptor Corrects the Impaired Mitochondrial Energy Status Observed in Renal Polycystin-1 Knockdown Cells Modeling Autosomal Dominant Polycystic Kidney Disease

Mise

Ranieri

Centrone

et al. 2018

Front. Mol. Biosci.

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Autosomal Dominant Polycistic kidney Disease (ADPKD) is a renal channelopathy due to loss-of-function mutations in the PKD1 or PKD2 genes, encoding polycystin-1 (PC1) or polycystin-2 (PC2), respectively. PC1 is a large protein found predominantly on the plasma membrane where interacts with different proteins, including PC2. PC2 is a smaller integral membrane protein also expressed in intracellular organelles, acting as a non-selective cation channel permeable to calcium. Both PC1 and PC2 are also localized to the primary cilium of renal epithelial cells serving as mechanosensor that controls calcium influx through the plasma membrane and regulates intracellular calcium release from the endoplasmic reticulum. The mechanisms by which PC1/2 dysfunction leads to ADPKD needs still to be clarified. We have recently reported that selective Calcium-Sensing Receptor (CaSR) activation in human conditionally immortalized Proximal Tubular Epithelial cells deficient for PC1 (ciPTEC-PC1KD), deriving from urine sediments reduces intracellular cAMP and mTOR activity, and increases intracellular calcium reversing the principal ADPKD dysregulations. Reduced cellular free calcium found in ADPKD can, on the other hand, affect mitochondrial function and ATP production and, interestingly, a relationship between mitochondria and renal polycystic diseases have been suggested. By using ciPTEC-PC1KD as experimental tool modeling of ADPKD, we show here that, compared with wild type cells, ciPTEC-PC1KD have significantly lower mitochondrial calcium levels associated with a severe deficit in mitochondrial ATP production, secondary to a multilevel impairment of oxidative phosphorylation. Notably, selective CaSR activation with the calcimimetic NPS-R568 increases mitochondrial calcium content close to the levels found in resting wild type cells, and fully recovers the cell energy deficit associated to the PC1 channel disruption. Treatment of ciPTEC-PC1KD with 2-APB, an IP3R inhibitor, prevented the rescue of bioenergetics deficit induced by CaSR activation supporting a critical role of IP3Rs in driving ER-to-mitochondria Ca2+ shuttle. Together these data indicate that, besides reversing the principal dysregulations considered the most proximal events in ADPKD pathogenesis, selective CaSR activation in PKD1 deficient cells restores altered mitochondrial function that, in ADPKD, is known to facilitate cyst formation. These findings identify CaSR as a potential therapeutic target.

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“…Furthermore, in vitro studies showed that mutation of a STAT6 binding motif abolished the IL4/IL13 effects on pendrin promoter activity [24]. Studies from our group showed that stimulation with forskolin or treatment with IL13 increased the abundance of pendrin in the plasma membrane of bronchial NCI-H292 cells [42,43], which may contribute to modulating the intracellular pH [42].…”

Section: Pendrin Expression and Function In The Airwaysmentioning

confidence: 94%

Functional interplay between CFTR and pendrin: physiological and pathophysiological relevance

Tamma¹,

Dossena²

2022

Front. Biosci. (Landmark Ed)

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The transport of chloride and bicarbonate across epithelia controls the pH and volume of the intracellular and luminal fluids, as well as the systemic pH and vascular volume. The anion exchanger pendrin (SLC26A4) and the cystic fibrosis transmembrane conductance regulator (CFTR) channel are expressed in the apical membrane of epithelial cells of various organs and tissues, including the airways, kidney, thyroid, and inner ear. While pendrin drives chloride reabsorption and bicarbonate, thiocyanate or iodide secretion within the apical compartment, CFTR represents a pathway for the apical efflux of chloride, bicarbonate, and possibly iodide. In the airways, pendrin and CFTR seems to be involved in alkalinization of the apical fluid via bicarbonate secretion, especially during inflammation, while CFTR also controls the volume of the apical fluid via a cAMP-dependent chloride secretion, which is stimulated by pendrin. In the kidney, pendrin is expressed in the cortical collecting duct and connecting tubule and co-localizes with CFTR in the apical membrane of β intercalated cells. Bicarbonate secretion occurs via pendrin, which also drives chloride reabsorption. A functional CFTR is required for pendrin activity. Whether CFTR stimulates pendrin via a direct molecular interaction or other mechanisms, or simply provides a pathway for chloride recycling across the apical membrane remains to be established. In the thyroid, CFTR and pendrin might have overlapping functions in driving the apical flux of iodide within the follicular lumen. In other organs, including the inner ear, the possible functional interplay between pendrin and CFTR needs to be explored.

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A FRET-Based Approach for Quantitative Evaluation of Forskolin-Induced Pendrin Trafficking at the Plasma Membrane in Bronchial NCI H292 Cells

Cited by 8 publications

References 43 publications

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment

The V2 receptor antagonist tolvaptan raises cytosolic calcium and prevents AQP2 trafficking and function: an in vitro and in vivo assessment

Activation of the Calcium-Sensing Receptor Corrects the Impaired Mitochondrial Energy Status Observed in Renal Polycystin-1 Knockdown Cells Modeling Autosomal Dominant Polycystic Kidney Disease

Functional interplay between CFTR and pendrin: physiological and pathophysiological relevance

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