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.

Human airway epithelial cells express β-adrenergic receptors (β-ARs), which regulate mucociliary clearance by stimulating transepithelial anion transport and ciliary beat frequency. Previous studies using airway epithelial cells showed that stimulation with isoproterenol increased cell migration and wound repair by a cAMP-dependent mechanism. In the present study, impedance-sensing arrays were used to measure cell migration and epithelial restitution following wounding of confluent normal human bronchial epithelial (NHBE) and Calu-3 cells by electroporation. Stimulation with epinephrine or the β2-AR-selective agonist salbutamol significantly delayed wound closure and reduced the mean surface area of lamellipodia protruding into the wound. Treatment with the β-AR bias agonist carvedilol or isoetharine also produced a delay in epithelial restitution similar in magnitude to epinephrine and salbutamol. Measurements of extracellular signal-regulated kinase phosphorylation following salbutamol or carvedilol stimulation showed no significant change in the level of phosphorylation compared with untreated control cells. However, inhibition of protein phosphatase 2A activity completely blocked the delay in wound closure produced by β-AR agonists. In Calu-3 cells, where CFTR expression was inhibited by RNAi, salbutamol did not inhibit wound repair, suggesting that β-AR agonist stimulation and loss of CFTR function share a common pathway leading to inhibition of epithelial repair. Confocal images of the basal membrane of Calu-3 cells labeled with anti-β1-integrin (clone HUTS-4) antibody showed that treatment with epinephrine or carvedilol reduced the level of activated integrin in the membrane. These findings suggest that treatment with β-AR agonists delays airway epithelial repair by a G protein- and cAMP-independent mechanism involving protein phosphatase 2A and a reduction in β1-integrin activation in the basal membrane.

Background: Cystic fibrosis (CF) is a genetic disease affecting multiple organ systems of the body and is characterized by mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Previous work has shown that a single dose of aβ-agonist increases cardiac output (Q) and stroke volume (SV) and decreases systemic vascular resistance (SVR) in healthy subjects. This effect is attenuated in patients with CF; however, the mechanism is unknown. Potential explanations for this decreased cardiovascular response to a β-agonist in CF include inherent cardiovascular deficits secondary to the CFTR mutation, receptor desensitization from prolonged β-agonist use as part of clinical care, or inhibited drug delivery to the bloodstream due to mucus buildup in the lungs. This study sought to determine the effects of endogenous epinephrine (EPI) and norepinephrine (NE) on cardiovascular function in CF and to evaluate the relationship between cardiovascular function and CFTR F508del mutation. Methods: A total of 19 patients with CF and 31 healthy control subjects completed an assessment of Q (C 2 H 2 rebreathing), SV (calculated from Q and heart rate [HR]), Q and SV indexed to body surface area (BSA, QI, and SVI, respectively), SVR (through assessment of Q and mean arterial blood pressure [MAP]), and HR (from 12-lead electrocardiogram [ECG]) at rest along with plasma measures of EPI and NE. We compared subjects by variables of cardiovascular function relative to EPI and NE, and also based on genetic variants of the F508del mutation (homozygous deletion for F508del, heterozygous deletion for F508del, or no deletion of F508del). Results: Cystic fibrosis patients demonstrated significantly lower BSA (CF = 1.71 ± 0.05 m 2 vs healthy = 1.84 ± 0.04 m 2 , P = .03) and SVI (CF = 30.6 ± 2.5 mL/beat/m 2 vs healthy = 39.9 ± 2.5 mL/beat/m 2 , P = .02) when compared with healthy subjects. Cystic fibrosis patients also demonstrated lower Q (CF = 4.58 ± 0.36 L/min vs healthy = 5.71 ± 0.32 L/min, P = .03) and SV (CF = 54 ± 5.5 mL/beat vs healthy = 73.3 ± 4.5 mL/beat, P = .01), and a higher HR (CF = 93.2 ± 3.9 bpm vs healthy = 80.5 ± 2.7 bpm, P < .01) and SVR (CF = 2082 ± 156 dynes*s/cm −5 vs healthy = 1616 ± 74 dynes*s/cm −5 , P = .01) compared with healthy subjects. Furthermore, CF patients demonstrated a lower SV ( P < .01) corrected for NE when compared with healthy subjects. No significant differences were seen in HR or Q relative to NE, or SVR relative to EPI. Differences were seen in SV (F (2,14) = 7.982, ...

Carvedilol functions as a nonselective ␤-adrenergic receptor (AR)/␣1-AR antagonist that is used for treatment of hypertension and heart failure. Carvedilol has been shown to function as an inverse agonist, inhibiting G protein activation while stimulating ␤-arrestin-dependent signaling and inducing receptor desensitization. In the present study, short-circuit current (Isc) measurements using human airway epithelial cells revealed that, unlike ␤-AR agonists, which increase Isc, carvedilol decreases basal and 8-(4-chlorophenylthio)adenosine 3=,5=-cyclic monophosphatestimulated current. The decrease in Isc resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR). The carvedilol effect was abolished by pretreatment with the ␤2-AR antagonist ICI-118551, but not the ␤1-AR antagonist atenolol or the ␣ 1-AR antagonist prazosin, indicating that its inhibitory effect on Isc was mediated through interactions with apical ␤2-ARs. However, the carvedilol effect was blocked by pretreatment with the microtubuledisrupting compound nocodazole. Furthermore, immunocytochemistry experiments and measurements of apical CFTR expression by Western blot analysis of biotinylated membranes revealed a decrease in the level of CFTR protein in monolayers treated with carvedilol but no significant change in monolayers treated with epinephrine. These results demonstrate that carvedilol binding to apical ␤ 2-ARs inhibited CFTR current and transepithelial anion secretion by a mechanism involving a decrease in channel expression in the apical membrane. inverse agonists; bias ligands; ␤-arrestin signaling; carvedilol

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