17β-Estradiol inhibits Ca2+-dependent homeostasis of airway surface liquid volume in human cystic fibrosis airway epithelia

Normal airways homeostatically regulate the volume of airway surface liquid (ASL) through both cAMP- and Ca²⁺-dependent regulation of ion and water transport. In cystic fibrosis (CF), a genetic defect causes a lack of cAMP-regulated CFTR activity, leading to diminished Cl^– and water secretion from airway epithelial cells and subsequent mucus plugging, which serves as the focus for infections. Females with CF exhibit reduced survival compared with males with CF, although the mechanisms underlying this sex-related disadvantage are unknown. Despite the lack of CFTR, CF airways retain a limited capability to regulate ASL volume, as breathing-induced ATP release activates salvage purinergic pathways that raise intracellular Ca²⁺ concentration to stimulate an alternate pathway to Cl^– secretion. We hypothesized that estrogen might affect this pathway by reducing the ability of airway epithelia to respond appropriately to nucleotides. We found that uridine triphosphate–mediated (UTP-mediated) Cl^– secretion was reduced during the periovulatory estrogen maxima in both women with CF and normal, healthy women. Estrogen also inhibited Ca²⁺ signaling and ASL volume homeostasis in non-CF and CF airway epithelia by attenuating Ca²⁺ influx. This inhibition of Ca²⁺ signaling was prevented and even potentiated by estrogen antagonists such as tamoxifen, suggesting that antiestrogens may be beneficial in the treatment of CF lung disease because they increase Cl^– secretion in the airways.     

Inflammatory cytokines TNF-α and IL-17 enhance the efficacy of cystic fibrosis transmembrane conductance regulator modulators

Without cystic fibrosis transmembrane conductance regulator–mediated (CFTR-mediated) HCO₃^– secretion, airway epithelia of newborns with cystic fibrosis (CF) produce an abnormally acidic airway surface liquid (ASL), and the decreased pH impairs respiratory host defenses. However, within a few months of birth, ASL pH increases to match that in non-CF airways. Although the physiological basis for the increase is unknown, this time course matches the development of inflammation in CF airways. To learn whether inflammation alters CF ASL pH, we treated CF epithelia with TNF-α and IL-17 (TNF-α+IL-17), 2 inflammatory cytokines that are elevated in CF airways. TNF-α+IL-17 markedly increased ASL pH by upregulating pendrin, an apical Cl^–/HCO₃^– exchanger. Moreover, when CF epithelia were exposed to TNF-α+IL-17, clinically approved CFTR modulators further alkalinized ASL pH. As predicted by these results, in vivo data revealed a positive correlation between airway inflammation and CFTR modulator–induced improvement in lung function. These findings suggest that inflammation is a key regulator of HCO₃^– secretion in CF airways. Thus, they explain earlier observations that ASL pH increases after birth and indicate that, for similar levels of inflammation, the pH of CF ASL is abnormally acidic. These results also suggest that a non-cell-autonomous mechanism, airway inflammation, is an important determinant of the response to CFTR modulators.     

Synergistic airway gland mucus secretion in response to vasoactive intestinal peptide and carbachol is lost in cystic fibrosis

Cystic fibrosis (CF) is caused by dysfunction of the CF transmembrane conductance regulator (CFTR), an anion channel whose dysfunction leads to chronic bacterial and fungal airway infections via a pathophysiological cascade that is incompletely understood. Airway glands, which produce most airway mucus, do so in response to both acetylcholine (ACh) and vasoactive intestinal peptide (VIP). CF glands fail to secrete mucus in response to VIP, but do so in response to ACh. Because vagal cholinergic pathways still elicit strong gland mucus secretion in CF subjects, it is unclear whether VIP-stimulated, CFTR-dependent gland secretion participates in innate defense. It was recently hypothesized that airway intrinsic neurons, which express abundant VIP and ACh, are normally active and stimulate low-level gland mucus secretion that is a component of innate mucosal defenses. Here we show that low levels of VIP and ACh produced significant mucus secretion in human glands via strong synergistic interactions; synergy was lost in glands of CF patients. VIP/ACh synergy also existed in pig glands, where it was CFTR dependent, mediated by both Cl(-) and HCO(3) (-), and clotrimazole sensitive. Loss of "housekeeping" gland mucus secretion in CF, in combination with demonstrated defects in surface epithelia, may play a role in the vulnerability of CF airways to bacterial infections.     

Normal mouse intestinal mucus release requires cystic fibrosis transmembrane regulator–dependent bicarbonate secretion

The mechanisms underlying mucus-associated pathologies in cystic fibrosis (CF) remain obscure. However, recent studies indicate that CF transmembrane conductance regulator (CFTR) is required for bicarbonate (HCO₃^–) transport and that HCO₃^– is critical for normal mucus formation. We therefore investigated the role of HCO₃^– in mucus secretion using mouse small intestine segments ex vivo. Basal rates of mucus release in the presence or absence of HCO₃^– were similar. However, in the absence of HCO₃^–, mucus release stimulated by either PGE₂ or 5-hydroxytryptamine (5-HT) was approximately half that stimulated by these molecules in the presence of HCO₃^–. Inhibition of HCO₃^– and fluid transport markedly reduced stimulated mucus release. However, neither absence of HCO₃^– nor inhibition of HCO₃^– transport affected fluid secretion rates, indicating that the effect of HCO₃^– removal on mucus release was not due to decreased fluid secretion. In a mouse model of CF (mice homozygous for the most common human CFTR mutation), intestinal mucus release was minimal when stimulated with either PGE₂ or 5-HT in the presence or absence of HCO₃^–. These data suggest that normal mucus release requires concurrent HCO₃^– secretion and that the characteristically aggregated mucus observed in mucin-secreting organs in individuals with CF may be a consequence of defective HCO₃^– transport.     

Hyposecretion of fluid from tracheal submucosal glands of CFTR-deficient pigs

Cystic fibrosis (CF) results from mutations that disrupt CF transmembrane conductance regulator (CFTR), an anion channel found mainly in apical membranes of epithelial cells. CF leads to chronic infection of the airways with normally innocuous bacteria and fungi. Hypotheses to explain the pathophysiology of CF airways have been difficult to test because mouse models of CF do not develop human-like airway disease. The recent production of pigs lacking CFTR and pigs expressing the most common CF-causing CFTR mutant, ΔF508, provide another model that might help clarify the pathophysiology of CF airway disease. Here, we studied individual submucosal glands from 1-day-old piglets in situ in explanted tracheas, using optical methods to monitor mucus secretion rates from multiple glands in parallel. Secretion rates from control piglets (WT and CFTR^+/–) and piglets with CF-like disease (CFTR^–/– and CFTR^–/ΔF508) were measured under 5 conditions: unstimulated (to determine basal secretion), stimulated with forskolin, stimulated with carbachol, stimulated with substance P, and, as a test for synergy, stimulated with forskolin and a low concentration of carbachol. Glands from piglets with CF-like disease responded qualitatively to all agonists like glands from human patients with CF, producing virtually no fluid in response to stimulation with forskolin and substantially less in response to all other agonists except carbachol. These data are a step toward determining whether gland secretory defects contribute to CF airway disease.     

IRBIT coordinates epithelial fluid and HCO3– secretion by stimulating the transporters pNBC1 and CFTR in the murine pancreatic duct

Fluid and HCO₃^– secretion are vital functions of secretory epithelia. In most epithelia, this entails HCO₃^– entry at the basolateral membrane, mediated by the Na⁺-HCO₃^– cotransporter, pNBC1, and exit at the luminal membrane, mediated by a CFTR-SLC26 transporters complex. Here we report that the protein IRBIT (inositol-1,4,5-trisphosphate [IP₃] receptors binding protein released with IP₃), a previously identified activator of pNBC1, activates both the basolateral pNBC1 and the luminal CFTR to coordinate fluid and HCO₃^– secretion by the pancreatic duct. We used video microscopy and ion selective microelectrodes to measure fluid secretion and Cl^– and HCO₃^– concentrations in cultured murine sealed intralobular pancreatic ducts. Short interference RNA–mediated knockdown of IRBIT markedly inhibited ductal pNBC1 and CFTR activities, luminal Cl^– absorption and HCO₃^– secretion, and the associated fluid secretion. Single-channel measurements suggested that IRBIT regulated CFTR by reducing channel mean close time. Furthermore, expression of IRBIT constructs in HEK cells revealed that activation of pNBC1 required only the IRBIT PEST domain, while activation of CFTR required multiple IRBIT domains, suggesting that IRBIT activates these transporters by different mechanisms. These findings define IRBIT as a key coordinator of epithelial fluid and HCO₃^– secretion and may have implications to all CFTR-expressing epithelia and to cystic fibrosis.     

Protein kinase C‐δ mediates von Willebrand factor secretion from endothelial cells in response to vascular endothelial growth factor (VEGF) but not histamine

Summary. Background: Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of exocytosis in many secretory cell types. Objectives: We investigated the role of PKC and the interactions between PKC and Ca²⁺ signaling in both VEGF‐induced and histamine‐induced VWF secretion from human umbilical vein endothelial cells (HUVECs). Results: Several PKC inhibitors (staurosporine, Ro31‐8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF‐induced but not histamine‐induced VWF secretion. PKC‐α and novel PKCs (PKC‐δ, PKC‐ε, and PKC‐η), but not PKC‐β, are expressed in HUVECs. Both VEGF and histamine activate PKC‐δ. However, gene inactivation experiments using small interfering RNA indicate that PKC‐δ (but not PKC‐α) is involved in the regulation of VEGF‐induced but not histamine‐induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca²⁺ ([Ca²⁺]_c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF‐induced secretion is largely preserved when the rise in [Ca²⁺]_c is prevented by BAPTA‐AM. Conclusions: Our study identifies striking agonist specificities in signal–secretion coupling. Histamine‐induced secretion is dependent on [Ca²⁺]_c but not PKC, whereas VEGF‐induced secretion is largely dependent on PKC‐δ and significantly less on [Ca²⁺]_c. Our data firmly establish the key role of PKC‐δ in VEGF‐induced VWF release, but suggest that a third, VEGF‐specific, signaling intermediate is required as a PKC‐δ coactivator.     

Defective regulation of gap junctional coupling in cystic fibrosis pancreatic duct cells

The cystic fibrosis (CF) gene encodes a cAMP-gated Cl- channel (cystic fibrosis transmembrane conductance regulator [CFTR]) that mediates fluid transport across the luminal surfaces of a variety of epithelial cells. We have previously shown that gap junctional communication and Cl- secretion were concurrently regulated by cAMP in cells expressing CFTR. To determine whether intercellular communication and CFTR-dependent secretion are related, we have compared gap junctional coupling in a human pancreatic cell line harboring the DeltaF508 mutation in CFTR and in the same cell line in which the defect was corrected by transfection with wild-type CFTR. Both cell lines expressed connexin45 (Cx45), as evidenced by RT-PCR, immunocytochemistry, and dual patch-clamp recording. Exposure to agents that elevate intracellular cAMP or specifically activate protein kinase A evoked Cl- currents and markedly increased junctional conductance of CFTR-expressing pairs, but not in the parental cells. The latter effect, which was caused by an increase in single-channel activity but not in unitary conductance of Cx45 channels, was not prevented by exposing CFTR-expressing cells to a Cl- channel blocker. We conclude that expression of functional CFTR restored the cAMP-dependent regulation of junctional conductance in CF cells. Direct intercellular communication coordinates multicellular activity in tissues that are major targets of CF manifestations. Consequently, defective regulation of gap junction channels may contribute to the altered functions of tissues affected in CF.     

Phorbol ester‐evoked Ca2+signaling in human platelets is via autocrine activation of P2X1 receptors,not a novel non‐capacitative Ca2+entry

Summary. Background: Platelets are reported to possess a protein kinase C (PKC)‐dependent non‐capacitative Ca²⁺entry (NCCE) pathway. The phorbol ester, phorbol, 12‐myristate, 13‐acetate (PMA) has been suggested to stimulate platelet NCCE. Recently we demonstrated important roles in store‐operated Ca²⁺entry in human platelets for Na⁺/Ca²⁺ exchangers (NCXs) and autocrine signaling between platelets after dense granule secretion. As PMA evokes dense granule secretion, we have investigated the role of NCXs and autocrine signaling in PMA‐evoked Ca²⁺entry. Objectives: To investigate the roles of NCXs and dense granule secretion in PMA‐evoked Ca²⁺signaling in human platelets. Methods: Fura‐2‐ or sodium‐binding benzofuran isophthalate (SBFI)‐loaded platelets were used to monitor cytosolic Ca²⁺or Na⁺ concentrations. Dense granule secretion was monitored as ATP release using luciferin–luciferase. Results: The NCX inhibitors KB‐R7943 or SN‐6, and removal of extracellular Na⁺, significantly reduced PMA‐evoked Ca²⁺entry. PMA‐evoked dense granule secretion was almost abolished by pretreatment with the PKC inhibitor Ro‐31‐8220 and significantly slowed by KB‐R7943. The P_2X1 antagonists Ro‐0437626 or MRS‐2159, or desensitization of P_2X1 receptors by prior treatment with α,β‐Methylene‐ATP or omitting apyrase from the medium, reduced PMA‐evoked Ca²⁺entry. Ro‐0437626 or chelation of extracellular Ca²⁺ slowed but did not abolish PMA‐evoked ATP release, indicating that PMA‐evoked dense granule secretion does not require P_2X1 receptor activation but is accelerated by P_2X1‐mediated Ca²⁺entry. The presence of NCX3 in human platelets was demonstrated by Western blotting. Conclusion: PMA‐evoked Ca²⁺entry results from an NCX3‐dependent dense granule secretion and subsequent P_2X1 receptor activation by secreted ATP, rather than activation of a novel NCCE pathway.     

Cystic fibrosis and estrogens: a perfect storm

Irreversible destruction and widening of the airways due to acquired infections or genetic mutations as well as those of unknown cause are more severe in females. Differences between male and female anatomy, behavior, and hormonal state have been proposed to explain the increased incidence and severity in females with airway disease such as cystic fibrosis (CF); however, a mechanism to explain a sex-related difference has remained elusive. In this issue of the JCI, Coakley et al. report that elevations in the major estrogen hormone in humans — 17β-estradiol — reduce Ca²⁺-activated Cl^– secretion by airway epithelial cells in culture, thereby disrupting ion and water balance (see the related article beginning on page 4025). They measure a similar diminution of nasal epithelial Ca²⁺-activated Cl^– secretion in women with CF during the menstrual cycle phase at which 17β-estradiol level is at its highest. These data suggest that for about one week of a four-week menstrual cycle, women with CF will have a reduced ability to efficiently clear airway secretions, the buildup of which is a hallmark of CF. The authors suggest that these data warrant the testing of antiestrogen therapy in females with CF and propose an alternative avenue for CF therapeutic development.     

Antisense oligodeoxynucleotide to the cystic fibrosis transmembrane conductance regulator inhibits cyclic AMP-activated but not calcium-activated cell volume reduction in a human pancreatic duct cell line.

Cystic fibrosis (CF) is characterized by a defect in cAMP-regulated chloride channels in epithelial cells. The CF gene product CF transmembrane conductance regulator (CFTR) is expressed in the apical membrane of pancreatic duct cells, and mutant CFTR accounts for the pathology in the CF pancreas. PANC 1, a pancreatic duct cell line, has not been considered a good model for studying CFTR and pancreatic chloride transport because CFTR mRNA and protein are undetectable using standard methods. Using electronic cell sizing and cell volume reduction under isotonic conditions, PANC 1 cells were found to possess both cAMP and calcium-activated chloride conductances. Using CFTR antisense oligodeoxynucleotides, the cAMP-activated conductance could be specifically inhibited in a concentration- and time-dependent manner. These findings demonstrate that PANC 1 cells express CFTR and a CFTR-independent calcium-activated chloride channel. With electronic cell sizing and CFTR antisense oligodeoxynucleotides, PANC 1 cells can provide an ideal system for the study of pancreatic duct cell physiology and pathophysiology with respect to the role of CFTR in the pancreas. These findings also suggest that antisense oligodeoxynucleotides may provide a more sensitive yet highly specific means of detecting low levels of expression of CFTR than currently available.     

ARC 118925XX stimulates cation influx in bEND.3 endothelial cells

In a previous publication when we studied the purinergic receptor with which ATP interacted in mouse brain bEND.3 endothelial cells, we observed addition of 3 μm ARC 118925XX (ARC; selective P2Y₂ antagonist) strongly suppressed ATP‐triggered Ca²⁺ release, suggesting the response was mediated via P2Y₂ receptors. We here report ARC unexpectedly promoted substantial Ca²⁺ influx even when ATP‐triggered Ca²⁺ release was largely inhibited. Since this large Ca²⁺ influx may have important pharmacological significance, we proceeded to investigate its mechanism. ARC did not trigger intracellular Ca²⁺ release thus suggesting Ca²⁺ influx triggered by ARC was not store‐operated. ARC‐triggered Ca²⁺ influx could be blocked by 1 mm Ni²⁺, a general Ca²⁺ channel blocker, but not by SK&F 96365, a nonselective TRP channel blocker. Unexpectedly, ARC promoted influx of Na⁺ and La³⁺, but not Mn²⁺. This is a surprising finding, since Mn²⁺ is conventionally used as a Ca²⁺ surrogate ion (as it permeates Ca²⁺ channel), and La³⁺ is classically used as a potent Ca²⁺ channel antagonist. Electrophysiological examination showed ARC did not stimulate any cation currents. Therefore, ARC opened, rather than a cation channel pore, an unidentified Ca²⁺ influx pathway which was Na⁺‐ and La³⁺‐permeable but Mn²⁺‐impermeable.     

Disease phenotype of a ferret CFTR-knockout model of cystic fibrosis

Cystic fibrosis (CF) is a recessive disease that affects multiple organs. It is caused by mutations in CFTR. Animal modeling of this disease has been challenging, with species- and strain-specific differences in organ biology and CFTR function influencing the emergence of disease pathology. Here, we report the phenotype of a CFTR-knockout ferret model of CF. Neonatal CFTR-knockout ferrets demonstrated many of the characteristics of human CF disease, including defective airway chloride transport and submucosal gland fluid secretion; variably penetrant meconium ileus (MI); pancreatic, liver, and vas deferens disease; and a predisposition to lung infection in the early postnatal period. Severe malabsorption by the gastrointestinal (GI) tract was the primary cause of death in CFTR-knockout kits that escaped MI. Elevated liver function tests in CFTR-knockout kits were corrected by oral administration of ursodeoxycholic acid, and the addition of an oral proton-pump inhibitor improved weight gain and survival. To overcome the limitations imposed by the severe intestinal phenotype, we cloned 4 gut-corrected transgenic CFTR-knockout kits that expressed ferret CFTR specifically in the intestine. One clone passed feces normally and demonstrated no detectable ferret CFTR expression in the lung or liver. The animals described in this study are likely to be useful tools for dissecting CF disease pathogenesis and developing treatments.     

Defective fluid transport by cystic fibrosis airway epithelia.

Cystic fibrosis (CF) airway epithelia exhibit defective transepithelial electrolyte transport: cAMP-stimulated Cl- secretion is abolished because of the loss of apical membrane cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, and amiloride-sensitive Na+ absorption is increased two- to threefold because of increased amiloride-sensitive apical Na+ permeability. These abnormalities are thought to alter respiratory tract fluid, thereby contributing to airway disease, the major source of mortality in this genetic disease. However, the underlying hypothesis, that fluid transport is abnormal in CF airway epithelia, has not been tested. Most conjecture about fluid transport is based on measurements of Na+ and Cl- transport performed under short circuit conditions in Ussing chambers. But such studies differ from in vivo conditions in that transepithelial voltage and mucosal fluid composition are held constant. Therefore, we measured fluid transport and mucosal electrolyte composition in primary cultures of CF airway epithelia without holding transepithelial voltage and ion concentration gradients at zero. In normal epithelia, cAMP agonists plus amiloride stimulated NaCl and fluid secretion. In CF epithelia, cAMP agonists failed to stimulate fluid or electrolyte secretion, changes consistent with the loss of CFTR Cl- channels. But in striking contrast to predictions based on Ussing chamber studies, CF epithelia absorbed fluid at a rate no greater than normal epithelia. Moreover, amiloride, which inhibits Na+ channels, failed to inhibit fluid absorption by CF epithelia. These results have important implications for understanding the pathogenesis of CF airway disease and for the design and evaluation of therapy.     

Thyrotropin-releasing Hormone Stimulation of Prolactin Release from Clonal Rat Pituitary Cells: EVIDENCE FOR ACTION INDEPENDENT OF EXTRACELLULAR CALCIUM

Thyrotropin-releasing hormone (TRH) stimulates prolactin release and ⁴⁵Ca²⁺ efflux from GH₃ cells, a clonal strain of rat pituitary cells. Elevation of extracellular K⁺ also induces prolactin release and increases ⁴⁵Ca²⁺ efflux from these cells. In this report, we distinguish between TRH and high K⁺ as secretagogues and show that TRH-induced release of prolactin and ⁴⁵Ca²⁺ is independent of the extracellular Ca²⁺ concentration, but the effect of high K⁺ on prolactin release and ⁴⁵Ca²⁺ efflux is dependent on the concentration of Ca²⁺ in the medium. The increment in ⁴⁵Ca²⁺ efflux induced by 50 mM K⁺ during perifusion was reduced in a concentration-dependent manner by lowering extracellular Ca²⁺ from 1,500 to 0.02 μM (by adding EGTA), whereas 1 μM TRH enhanced ⁴⁵Ca²⁺ efflux similarly over the entire range of extracellular Ca²⁺ concentrations. Although 50 mM K⁺ caused release of 150 ng prolactin from 40 × 10⁶ GH₃ cells exposed to 1,500 μM Ca²⁺ (control), reduction of extracellular Ca²⁺ to 2.8 μM decreased prolactin release caused by high K⁺ to <3% of controls and no prolactin release was detected after exposure to 50 mM K⁺ in medium with 0.02 μM free Ca²⁺. In contrast, TRH caused release of 64 ng of prolactin from 40 × 10⁶ GH₃ cells exposed to medium with 1,500 μM Ca²⁺, and release caused by TRH was still 50 and 35% of control in medium with 2.8 and 0.02 μM Ca²⁺, respectively. Furthermore, TRH transiently increased by 10-fold the fractional efflux of ⁴⁵Ca²⁺ from GH₃ cells in static incubations with 1,500 or 3.5 μM Ca²⁺, hereby confirming that the enhanced ⁴⁵Ca²⁺ efflux caused by TRH in both low and high Ca²⁺ medium was not an artifact of the perifusion system.     

Modulation of Intracellular Ca Concentration in Brain Microvascular Endothelial Cells in vitro by Acoustic Cavitation

Localized delivery of therapeutic agents through the blood–brain barrier (BBB) is a clinically significant task that remains challenging. Ultrasound (US) application after intravenous administration of microbubbles has been shown to generate localized BBB opening in animal models but the detailed mechanisms are not yet fully described. The current study investigates the effects of US-stimulated microbubbles on in vitro murine brain microvascular endothelial (bEnd.3) cells by monitoring sonoporation and changes in intracellular calcium concentration ([Ca²⁺]_i) using real-time fluorescence and high-speed brightfield microscopy. Cells seeded in microchannels were exposed to a single US pulse (1.25 MHz, 10 cycles, 0.24 MPa peak negative pressure) in the presence of Definity™ microbubbles and extracellular calcium concentration [Ca²⁺]_o = 0.9 mM. Disruption of the cell membrane was assessed using propidium iodide (PI) and change in the [Ca²⁺]_i was measured using fura-2. Cells adjacent to a microbubble exhibited immediate [Ca²⁺]_i changes after US pulse with and without PI uptake and the [Ca²⁺]_i changes were twice as large in cells with PI uptake. Cell viability assays showed that sonoporated cells could survive with modulation of [Ca²⁺]_i and uptake of PI. Cells located near sonoporated cells were observed to exhibit changes in [Ca²⁺]_i that were delayed from the time of US application and without PI uptake. These results demonstrate that US-stimulated microbubbles not only directly cause changes in [Ca²⁺]_i in brain endothelial cells in addition to sonoporation but also generate [Ca²⁺]_i transients in cells not directly interacting with microbubbles, thereby affecting cells in larger regions beyond the cells in contact with microbubbles. (E-mail: cxdeng@umich.edu)     

Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.     

Enhanced transient receptor potential channel‐mediated Ca2+ influx in the cells with phospholipase C‐δ1 overexpression: its possible role in coronary artery spasm

We reported that coronary spasm was induced in the transgenic mice with the increased phospholipase C (PLC)‐δ1 activity. We investigated the effect of enhanced PLC‐δ1 on Ca²⁺ influx and its underlying mechanisms. We used human embryonic kidney (HEK)‐293 and coronary arteries smooth muscle cells (CASMC). Intracellular free Ca²⁺ concentration ([Ca²⁺]_i; nm ) was measured by fura‐2, and Ca²⁺ influx was evaluated by the increase in [Ca²⁺]_i after addition of extracellular Ca²⁺. Acetylcholine (ACh) was used to induce Ca²⁺ influx. ACh‐induced peak Ca²⁺ influx was 19 ± 3 in control HEK‐293 cells and 71 ± 8 in the cells with PLC‐δ1 overexpression (P < 0.05 between two groups). Nifedipine partially suppressed this Ca²⁺ influx, whereas either 2‐APB or knockdown of classical transient receptor potential channel 6 (TRPC6) blocked this Ca²⁺ influx. In the human CASMC, ACh‐induced peak Ca²⁺ influx was 29 ± 6 in the control and was increased to 45 ± 16 by PLC‐δ1 overexpression (P < 0.05). Like HEK‐293 cells, pretreatment with nifedipine partially suppressed Ca²⁺ influx, whereas either 2‐APB or knockdown of TRPC6 blocked it. ACh‐induced Ca²⁺ influx was enhanced by PLC‐δ1 overexpression, and was blocked partially by nifedipine and completely by 2‐APB. TRPC‐mediated Ca²⁺ influx may be related to the enhanced Ca²⁺ influx in PLC‐δ1 overexpression.     

Vasoactive intestinal peptide increases cystic fibrosis transmembrane conductance regulator levels in the apical membrane of Calu-3 cells through a protein kinase C-dependent mechanism

Noncholinergic neurons contribute to innate airway defenses by releasing vasoactive intestinal peptides (VIP), which stimulates the submucosal glands to produce a bicarbonate-rich fluid containing mucins and antimicrobial factors. VIP elevates cAMP and activates cystic fibrosis transmembrane conductance regulator (CFTR) channels; however, its effects on surface expression have not been investigated. We studied CFTR levels in the apical membrane of polarized Calu-3 cell monolayers, a widely used model for submucosal gland serous cells. Biotinylation during VIP exposure revealed a significant increase in apical CFTR within 10 min, which reached a maximal 3.3-fold increase after 30 min. Total CFTR content of cell lysates was not altered during this time period; therefore, the increase in surface CFTR reflects redistribution from intracellular pools. Internalization assays revealed that apical accumulation was due, at least in part, to a reduction in the rate of CFTR endocytosis. VIP-induced accumulation of apical CFTR was mimicked by phorbol ester but not by forskolin, and it was blocked by the protein kinase (PK)C inhibitors bisindolylmaleimide X (BisX) or chelerythrine chloride but not by the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89). Increases in surface expression were paralleled by enhanced iodide effluxes during cAMP stimulation. BisX inhibition of VIP responses was abrogated when monolayers were pretreated with tannic acid to inhibit endosome recycling. Thus, PKC increases the surface expression of CFTR channels in addition to potentiating their responsiveness to PKA phosphorylation. Integrated regulation through multiple signaling pathways may be a common feature of VIP and other physiological secretagogues.     

Calcium influx through L-type CaV1.2 Ca2+ channels regulates mandibular development

The identification of a gain-of-function mutation in CACNA1C as the cause of Timothy Syndrome (TS), a rare disorder characterized by cardiac arrhythmias and syndactyly, highlighted unexpected roles for the L-type voltage-gated Ca²⁺ channel Ca_V1.2 in nonexcitable cells. How abnormal Ca²⁺ influx through Ca_V1.2 underlies phenotypes such as the accompanying syndactyly or craniofacial abnormalities in the majority of affected individuals is not readily explained by established Ca_V1.2 roles. Here, we show that Ca_V1.2 is expressed in the first and second pharyngeal arches within the subset of cells that give rise to jaw primordia. Gain-of-function and loss-of-function studies in mouse, in concert with knockdown/rescue and pharmacological approaches in zebrafish, demonstrated that Ca²⁺ influx through Ca_V1.2 regulates jaw development. Cranial neural crest migration was unaffected by Ca_V1.2 knockdown, suggesting a role for Ca_V1.2 later in development. Focusing on the mandible, we observed that cellular hypertrophy and hyperplasia depended upon Ca²⁺ signals through Ca_V1.2, including those that activated the calcineurin signaling pathway. Together, these results provide new insights into the role of voltage-gated Ca²⁺ channels in nonexcitable cells during development.