No abstract

1 Apical ATP, UTP and UDP evoked transient increases in short circuit current (I SC , a direct measure of transepithelial ion transport) in con¯uent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors.

The present study investigated the involvement of Na+-HCO3- cotransporter in mediating cAMP-stimulated HCO3- secretion across the cultured mouse endometrial epithelium using the short-circuit current (I(SC)) technique and intracellular pH measurement. Forskolin stimulated a rise in the I(SC), 55.6% and 52.1% of which could be reduced by the removal of extracellular Cl- or by eliminating the contribution of Cl- secretion by bumetanide, an inhibitor of Na+-K+-2Cl- cotransporter, respectively. More than 80% reduction in the forskolin-induced I(SC) was obtained when both Cl- and HCO3- in the bath were removed or in HCO3--free solution with bumetanide, indicating that the I(SC) depended on both Cl- and HCO3-. The presence of the Na+ channel-blocker amiloride in the apical solution did not reduce the forskolin-induced I(SC); however, the I(SC) could be abolished by removing Na+ from the bathing solution, suggesting that the Cl-- and HCO3--dependent I(SC) was also dependent on basolateral Na+. The forskolin-stimulated I(SC) could be reduced 43.6% by removal of HCO3- and 47.9% by a Na+-HCO3--cotransporter inhibitor, dihydrogen-4,4'-didsothiocyanostilbene-2,2'-disulfonic acid (H2DIDS). The inhibitory effect of H2DIDS was observed in Cl--free solution, but not when HCO3- was removed, thus confirming its effect on HCO3--dependent transport. Intracellular pH measurements demonstrated that the recovery from cellular acidification depended on the presence of both basolateral Na+ and HCO3-, further indicating the involvement of Na+-HCO3- cotransporter. Reverse transcription-polymerase chain reaction experiments confirmed the expression of Na+-HCO3- cotransporter in the mouse endometrium. The results suggest that basolaterally located Na+-HCO3- cotransporter is involved in mediating cAMP-stimulated HCO3- secretion across the mouse endometrial epithelium.

Background/Aims: Carbon monoxide (CO) is an important gas produced endogenously by heme oxygenase (HO) that functions as an anti-inflammatory and in ion channel modulation, but the effects of CO on airway inflammation and ion transport remains unclear. Methods: The effect of CO on cell damage- and nucleotide-induced pro-inflammatory cytokine release in primary human bronchial epithelia cells (HBE) and in the 16HBE14o- human bronchial epithelial cell line were investigated. The effects of CO on calcium- and cAMP-dependent chloride (Cl^-) secretion were examined using a technique that allowed the simultaneous measurement and quantification of real-time changes in signalling molecules (cAMP and Ca²⁺) and ion transport in a polarised epithelium. Results: CO suppressed the release of interleukin (IL)-6 and IL-8 and decreased the phosphorylation of ERK1/2 and NF-κB p65. Furthermore, CO inhibited UTP-induced increases in calcium and Cl^- secretion, and forskolin-induced increases in cAMP and Cl^- secretion. Conclusions: These findings suggest a novel anti-inflammatory role of CO in human bronchial epithelia via interactions with purinergic signalling pathways. Further, CO modulated both the Ca²⁺- and cAMP-dependent secretion of Cl^-.

Background/Aims: Carbon monoxide (CO) is an important autocrine/paracrine messenger involved in a variety of physiological and pathological processes. This study aimed to investigate the regulatory role of CO released by CO-releasing molecule-2 (CORM-2) in a P2Y receptor-mediated calcium-signaling pathway in the human bronchial epithelial cell line, 16HBE14o-. Methods: Intracellular calcium ([Ca²⁺]_i) was measured by fura-2 microspectrofluorimetry. D-myo-inositol-1-phosphate (IP₁) levels and cGMP-dependent protein kinase activity (PKG) were also quantified. Results: The exogenous application of CORM-2 increased both intracellular Ca²⁺ and IP₁, which are inhibited by U73122, a phospholipase C (PLC) inhibitor. In contrast, the P2Y₂/P2Y₄ receptor-mediated intracellular Ca²⁺ release and influx induced by UTP were inhibited in the presence of CORM-2. However, CORM-2 did not affect the store-operated Ca²⁺ entry (SOCE) induced by thapsigargin (Tg). Moreover, the inhibitory effect of CORM-2 on UTP-induced calcium increase could be attenuated by a soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), or a Protein Kinase G (PKG) inhibitor, KT5823, suggesting the involvement of sGC/PKG signaling in this process. Conclusion: CORM-2 serves a dual role in modulating [Ca²⁺]_i in 16HBE14o- cells. Thus, CO released by CORM-2 may act as a regulator of calcium homeostasis in human airway epithelia. These findings help further elucidate the function of CO in many physiological and pathological conditions.