Key points• The mechanisms of anion and fluid transport by airway submucosal glands are not well understood and may differ from those in surface epithelium.• The Calu-3 cell line is often used as a model for submucosal gland serous cells and has cAMP-stimulated fluid secretion; however, it does not actively transport chloride under short-circuit conditions.• In this study we show that fluid secretion requires chloride, bicarbonate and sodium, that chloride is the predominant anion in Calu-3 secretions, and that a large fraction of the basolateral chloride loading during cAMP stimulation occurs by Cl − /HCO 3 − exchange.• The results suggest a novel cellular model for anion and fluid secretion by Calu-3 and submucosal gland acinar cells Abstract Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl − and HCO 3 − secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (I sc ) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (I eq ) calculated under open-circuit conditions. I sc was equivalent to the HCO 3 − net flux measured using the pH-stat technique, whereas I eq was the sum of the Cl − and HCO 3 − net fluxes. I eq and HCO 3 − fluxes were increased by bafilomycin and ZnCl 2 , suggesting that some secreted HCO 3 − is neutralized by parallel electrogenic H + secretion. I eq and fluid secretion were dependent on the presence of both Na + and HCO 3 − . The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of I eq and HCO 3 − secretion, suggesting that HCO 3 − transport under these conditions requires catalysed synthesis of carbonic acid. Cl − was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl − and fluid transport was bumetanide-insensitive, suggesting basolateral Cl − loading by a sodium-potassium-chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO 3 − gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO 3 with Cl − , and the resulting HCO 3 − -dependent Cl − transport provides an osmotic driving force for fluid secretion.

Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl(-) secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (I(sc): thrombin, 21 +/- 2 microA; SLIGRL, 83 +/- 22 microA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca(2+)-chelating agent BAPTA-AM (50 microM) abolished the increase in I(sc) produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 microM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated I(sc) was observed. In addition, basolateral treatment with the PGE(2) receptor antagonist AH-6809 (25 microM) significantly inhibited the effects of SLIGRL on I(sc). QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca(2+)-activated KCNN4 K(+) channel, and the KCNQ1 K(+) channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl(-) conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl(-) secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl(-) secretion requires activation of CFTR and at least two distinct K(+) channels located in the basolateral membrane.

P2Y receptor regulation of anion secretion was investigated in porcine endometrial gland (PEG) epithelial cells. P2Y2, P2Y4, and P2Y6 receptors were detected in monolayers of PEG cells and immunocytochemistry indicated that P2Y4 receptors were located in the apical membrane. Apical membrane current measurements showed that Ca2+-dependent and PKC-dependent Cl- channels were activated following treatment with uridine triphosphate (UTP) (5 microM). Current-voltage relationships comparing calcium-dependent and PKC-dependent UTP responses under biionic conditions showed significant differences in selectivity between Cl-)and I- for the PKC-dependent conductance (P(I)/P(Cl) = 0.76), but not for Ca2+-dependent conductance (PI/P(Cl) = 1.02). The I-/Cl- permeability ratio for the PKC-dependent conductance was identical to that measured for 8-cpt cAMP. Furthermore, PKC stimulation using phorbol 12-myristate 13-acetate (PMA) activated an apical membrane Cl- conductance that was blocked by the CFTR selective inhibitor, CFTRinh-172. CFTR silencing, accomplished by stable expression of small hairpin RNAs (shRNA), blocked the PKC-activated conductance associated with UTP stimulation and provided definitive evidence of a role for CFTR in anion secretion. CFTR activation increased the initial magnitude of Cl- secretion, and provided a more sustained secretory response compared to conditions where only Ca2+-activated Cl- channels were activated by UTP. Measurements of [cAMP]i following UTP and PMA stimulation were not significantly different than untreated controls. Thus, these results demonstrate that UTP and PMA activation of CFTR occurs independently of increases in intracellular cAMP and extend the findings of earlier studies of CFTR regulation by PKC in Xenopus oocytes to a mammalian anion secreting epithelium.

Primary human mammary epithelial (HME) cells were immortalized by stable, constitutive expression of the catalytic subunit of human telomerase. Purinergic receptors were identified by RT-PCR and quantitative RT-PCR from mRNA isolated from primary and immortalized cells grown to confluence on membrane filters. Several subtypes of P2Y receptor mRNA were identified including P2Y(1), P2Y(2), P2Y(4), and P2Y(6) receptors. RT-PCR experiments also revealed expression of A(2b) adenosine receptor mRNA in primary and immortalized cells. Confluent monolayers of HME cells exhibited a basal short-circuit current (I(sc)) that was abolished by amiloride and benzamil. When monolayers were cultured in the presence of hydrocortisone, mRNA expression of Na(+) channel (ENaC) alpha-, beta-, and gamma-subunits increased approximately threefold compared with that in cells grown without hydrocortisone. In addition, basal benzamil-sensitive Na(+) transport was nearly twofold greater in hydrocortisone-treated monolayers. Stimulation with UTP, UDP, or adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS) produced increases in intracellular calcium concentration that were significantly reduced following pretreatment with the calcium-chelating agent BAPTA-AM. Concentration-response relationships indicated that the rank order of potency for these agonists was UTP > UDP > ATPgammaS. Basolateral stimulation with UTP produced a rapid but transient increase in I(sc) that was significantly reduced if cells were pretreated with BAPTA-AM or benzamil. Moreover, basolateral treatment with either charybdotoxin or clotrimazole significantly inhibited the initial UTP-dependent increase in I(sc) and eliminated the sustained current response. These results indicate that human mammary epithelial cells express multiple P2 receptor subtypes and that Ca(2+) mobilization evoked by P2Y receptor agonists stimulates Na(+) absorption by increasing the activity of Ca(2+)-activated K(+) channels located in the basolateral membrane.

Non-technical summary The epithelial cells lining the ducts of the human mammary gland are responsible for modifying sodium and potassium concentrations in milk by actively absorbing sodium from and secreting potassium into the ductal fluid. In the present study we show that adenosine triphosphate (ATP) and uridine triphosphate (UTP) can stimulate sodium absorption and potassium secretion by a mechanism that involves increasing intracellular calcium and activation of calcium-dependent potassium channels. We discovered that addition of ATP or UTP to the luminal surface stimulates potassium secretion, whereas addition of the same concentrations to the epithelial surface normally exposed to the blood produces an increase in sodium absorption. These results provide a better understanding of the signalling mechanisms that control the concentrations of sodium and potassium present in milk.Abstract Human mammary epithelial (HME) cells express several P2Y receptor subtypes located in both apical and basolateral membranes. Apical UTP or ATP-γ-S stimulation of monolayers mounted in Ussing chambers evoked a rapid, but transient decrease in short circuit current (I sc ), consistent with activation of an apical K + conductance. In contrast, basolateral P2Y receptor stimulation activated basolateral K + channels and increased transepithelial Na + absorption. Chelating intracellular Ca 2+ using the membrane-permeable compound BAPTA-AM, abolished the effects of purinoceptor activation on I sc . Apical pretreatment with charybdotoxin also blocked the I sc decrease by >90% and similar magnitudes of inhibition were observed with clotrimazole and TRAM-34. In contrast, iberiotoxin and apamin did not block the effects of apical P2Y receptor stimulation. Silencing the expression of K Ca 3.1 produced ∼70% inhibition of mRNA expression and a similar reduction in the effects of apical purinoceptor agonists on I sc . In addition, silencing P2Y 2 receptors reduced the level of P2Y 2 mRNA by 75% and blocked the effects of ATP-γ-S by 65%. These results suggest that P2Y 2 receptors mediate the effects of purinoceptor agonists on K + secretion by regulating the activity of K Ca 3.1 channels expressed in the apical membrane of HME cells. The results also indicate that release of ATP or UTP across the apical or basolateral membrane elicits qualitatively different effects on ion transport that may ultimately determine the [Na + ]/[K + ] composition of fluid within the mammary ductal network.