Abstract-Although the cationic inward rectifiers (Kir and hyperpolarization-activated I f channels) have been well characterized in cardiac myocytes, the expression and physiological role of anionic inward rectifiers in heart are unknown. In the present study, we report the functional and molecular identification of a novel chloride (Cl Ϫ ) inward rectifier (Cl.ir) in mammalian heart. Under conditions in which cationic inward rectifier channels were blocked, membrane hyperpolarization (Ϫ40 to Ϫ140 mV) activated an inwardly rectifying whole-cell current in mouse atrial and ventricular myocytes. Under isotonic conditions, the current activated slowly with a biexponential time course (time constants averaging 179.7Ϯ23.4 [meanϮSEM] and 2073.6Ϯ287.6 ms at Ϫ120 mV). Hypotonic cell swelling accelerated the activation and increased the current amplitude whereas hypertonic cell shrinkage inhibited the current. The inwardly rectifying current was carried by Cl Ϫ (I Cl.ir ) and had an anion permeability sequence of Cl Ϫ ϾI Ϫ Ͼ Ͼaspartate. I Cl.ir was blocked by 9-anthracene-carboxylic acid and cadmium but not by stilbene disulfonates and tamoxifen. A similar I Cl.ir was also observed in guinea pig cardiac myocytes. The properties of I Cl.ir are consistent with currents generated by expression of ClC-2 Cl Ϫ channels. Reverse transcription polymerase chain reaction and Northern blot analysis confirmed transcriptional expression of ClC-2 in both atrial and ventricular tissues and isolated myocytes of mouse and guinea pig hearts. These results indicate that a novel I Cl.ir is present in mammalian heart and support a potentially important role of ClC-2 channels in the regulation of cardiac electrical activity and cell volume under physiological and pathological conditions. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2000;86:e63-e71.)

P2‐purinoceptors couple extracellular ATP to the activation of a Cl− current (ICl,ATP) in heart. We studied the molecular mechanism and intracellular signalling pathways of ICl,ATP activation in mouse heart. Extracellular adenosine‐5′‐O‐(3‐thiotriphosphate) (ATPγS; 100 μM) activated ICl,ATP in both atrial and ventricular myocytes. A specific PKC inhibitor, bisindolylmaleimide blocked the effect of ATPγS while a PKC activator, phorbol 12,13‐dibutyrate (PDBu) activated a current with identical properties to ICl,ATP. Maximal activation of ICl,ATP by ATPγS or PDBu occluded further modulation by the other agonist, suggesting that they may activate the same population of Cl− channels. Isoprenaline increased ICl,ATP pre‐activated by ATPγS or PDBu, while isoprenaline or forskolin alone failed to activate any Cl− current in these myocytes. Adenosine 3′,5′‐cyclic monophosphothionate, a PKA inhibitor, prevented ATPγS or PDBu activation of ICl,ATP. Thus, ICl,ATP is regulated by dual intracellular phosphorylation pathways involving both PKA and PKC in a synergistic manner similar to cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channels. Glibenclamide (50 μM) significantly blocked ICl,ATP activated by ATPγS or by the CFTR channel activator, levamisole. The slope conductance of the unitary ICl,ATP in cell‐attached patches was 11·8 ± 0·3 pS, resembling the known properties of CFTR Cl− channels in cardiac myocytes. The reverse transcription polymerase chain reaction and Northern blot analysis revealed CFTR mRNA expression in mouse heart. We conclude that ICl,ATP in mouse heart is due to activation of CFTR Cl− channels through a novel intracellular signalling pathway involving purinergic activation of PKC and PKA.

ClC‐3 encodes a volume‐regulated Cl− channel (ICl,vol) in heart. We studied the regulation of native and recombinant cardiac ICl,vol by intracellular cyclic AMP (cAMPi). Symmetrical high Cl− concentrations were used to effectively separate outwardly rectifying ICl,vol from other non‐rectifying Cl− currents, such as the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca2+‐activated Cl− currents (ICl,CFTR and ICl,Ca, respectively), which are concomitantly expressed in cardiac myocytes. 8‐Bromo‐cyclic AMP (8‐Br‐cAMP) significantly inhibited ICl,vol in most guinea‐pig atrial myocytes. In ≈30 % of the atrial myocytes examined, 8‐Br‐cAMP increased macroscopic Cl− currents. However, the 8‐Br‐cAMP‐stimulated difference currents exhibited a linear current‐voltage (I–V) relation, consistent with activation of ICl,CFTR, not ICl,vol. In canine atrial myocytes, isoprenaline (1 μM) consistently reduced ICl,vol in Ca2+‐free hypotonic bath solutions with strong intracellular Ca2+ (Ca2+i) buffering. In Ca2+‐containing hypotonic bath solutions with weak Ca2+i buffering, however, isoprenaline increased net macroscopic Cl− currents. Isoprenaline‐stimulated difference currents were not outwardly rectifying, consistent with activation of ICl,Ca, not ICl,vol. In NIH/3T3 cells transfected with gpClC‐3 (the gene encoding ICl,vol), 8‐Br‐cAMP consistently inhibited ICl,ClC‐3. These effects were prevented by a protein kinase A (PKA) inhibitor, KT5720, or by mutation of a single consensus protein kinase C (PKC) phosphorylation site (S51A) on the N‐terminus of ClC‐3, which also mediates PKC inhibition of ICl,ClC‐3. We conclude that cAMPi causes inhibition of ICl,vol in mammalian heart due to cross‐phosphorylation of the same PKC consensus site on ClC‐3 by PKA. Our results suggest that contamination of macroscopic ICl,vol by ICl,CFTR and/or ICl,Ca may account for some of the inconsistent and controversial effects of cAMPi on ICl,vol previously reported in native cardiac myocytes.

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Vitamin D insufficiency/deficiency has been linked to an increased risk of preeclampsia. Impaired placental amino acid transport is suggested to contribute to abnormal fetal intrauterine growth in pregnancies complicated by preeclampsia. However, if vitamin D-regulated amino acid transporter is involved in the pathophysiologic mechanism of preeclampsia has not been clarified yet. The aberrant expression of key isoform of L-type amino acid transporter LAT1 was determined by western blot and immunohistochemistry in the placenta from normotensive and preeclamptic pregnancies. The role for vitamin D on placental LAT1 expression was investigated through the exposure of HTR-8/SVneo human trophoblast cells to the biologically active 1,25(OH)2D3 and the oxidative stress-inducer cobalt chloride (CoCl2). Our results showed that placental LAT1 expression was reduced in women with preeclampsia compared to normotensive pregnancies, which was associated with decreased expression of vitamin D receptor (VDR). 1,25(OH)2D3 significantly upregulated LAT1 expression in placental trophoblasts, and also prevented the decrease of mTOR activity under CoCl2-induced oxidative stress. siRNA targeting VDR significantly attenuated 1,25(OH)2D3-stimulated LAT1 expression and mTOR signaling activity. Moreover, treatment of rapamycin specifically inhibited the activity of mTOR signaling and resulted in decrease of LAT1 expression. In conclusion, LAT1 expression was downregulated in the placenta from women with preeclampsia. 1,25(OH)2D3/VDR could stimulate LAT1 expression, which was likely mediated by mTOR signaling in placental trophoblasts. Regulation on placental amino acid transport may be one of the mechanisms by which vitamin D affects fetal growth in preeclampsia.