Purinergic inhibition of Na+,K+,Cl− cotransport in C11-MDCK cells: Role of stress-activated protein kinases

Previously, we observed that sustained activation of P2Y1 leads to inhibition of Na+,K+,Cl− cotransport (NKCC) in C11 cells resembling intercalated cells from collecting ducts of the Madin-Darby canine kidney. This study examined the role of stress-activated protein kinases (SAPK) in NKCC inhibition triggered by purinergic receptors. Treatment of C11 cells with ATP led to sustained phosphorylation of SAPK such as JNK and p38. Activation of these kinases also occurred in anisomycin-treated cells. Surprisingly, we observed that compounds SP600125 and SB202190, known as potent inhibitors of JNK and p38 in cell-free systems, activated rather than inhibited phosphorylation of the kinases in C11 cells. Importantly, similarly to ATP, all the above-listed activators of JNK and p38 phosphorylation inhibited NKCC. Thus, our results suggest that activation of JNK and/or p38 contributes to NKCC suppression detected in intercalated-like cells from distal tubules after their exposure to P2Y1 agonists

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger-for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg2+ and/or H+ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP4- in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed

Therapeutic implications of Src independent calcium mobilization in diffuse large B-cell lymphoma

We report that 38% of primary large B-cell lymphoma (DLBCL) tested expressed active Src family kinases, which are targeted by dasatinib. The expression of active Src family of kinases (SFK) in primary DLBCL tumors correlated with unfavorable prognostic markers such as Ki67 and Mum1. Using four DLBCL cell lines we found that: (1) sensitivity to dasatinib (but not imatinib) varied 400-fold; (2) dasatinib resistance was associated with distinct signaling profiles downstream of BCR activation. In particular, although Src family kinase phosphorylation was inhibited by 100-150 nM dasatinib in all cell lines, this failed to inhibit BCR-mediated Blnk phosphorylation, calcium signaling and proliferation in a dasatinib resistant cell line

Imaging exocytosis of ATP-containing vesicles with TIRF microscopy in lung epithelial A549 cells

Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca2+-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells

Rapid non-genomic inhibition of ATP-induced Cl− secretion by dexamethasone in human bronchial epithelium

A non-genomic antisecretory role for dexamethasone at low concentrations (0.1 nm to1 μm) is described in monolayers of human bronchial epithelial cells in primary culture and in a continuous cell line (16HBE14o- cells). Dexamethasone produced a rapid decrease of [Ca2+]i (measured with fura-2 spectrofluorescence) to a new steady-state concentration. After 15 min exposure to dexamethasone (1 nm), [Ca2+]i was reduced by 32 ± 11 nm (n = 7, P < 0.0001) from a basal value of 213 ± 36 nm (n = 7). We have shown previously that aldosterone (1 nm) also produces a rapid fall in [Ca2+]i; however, after the decrease in [Ca2+]i induced by dexamethasone, subsequent addition of aldosterone did not produced any further lowering of [Ca2+]i. The rapid response to dexamethasone was insensitive to pretreatment with cycloheximide and unaffected by the glucocorticoid type II and mineralocorticoid receptor antagonists RU486 and spironolactone, respectively. The rapid [Ca2+]i decrease induced by dexamethasone was inhibited by the Ca2+-ATPase pump inhibitor thapsigargin (1 μm), the adenylate cyclase inhibitor MDL hydrochloride (500 μm) and the protein kinase A inhibitor Rp-adenosine 3′,5′-cyclic monophosphorothioate (200 μm), but was not affected by the protein kinase C inhibitor, chelerythrine chloride (0.1 μm). Treatment of 16HBE14o- cell monolayers with dexamethasone (1 nm) inhibited the large and transient [Ca2+]i increase induced by apical exposure to ATP (10−4m). Dexamethasone (1 nm) also reduced by 30 % the Ca2+-dependant Cl− secretion induced by apical exposure to ATP (measured as the Cl−-sensitive short-circuit current across monolayers mounted in Ussing chambers). Our results demonstrate, for the first time, that dexamethasone at low concentrations inhibits Cl− secretion in human bronchial epithelial cells. The rapid inhibition of Cl− secretion induced by the synthetic glucocorticoid is associated with a rapid decrease in [Ca2+]i via a non-genomic mechanism that does not involve the classical glucocorticoid or mineralocorticoid receptor. Rather, it is a result of rapid non-genomic stimulation of thapsigargin-sensitive Ca2+-ATPase, via adenylate cyclase and protein kinase A signalling