Disappearance of Hypoxic Pulmonary Vasoconstriction and O2-Sensitive Nonselective Cationic Current in Arterial Myocytes of Rats Under Ambient Hypoxia

Acute hypoxia induces contraction of pulmonary artery (PA) to protect ventilation/perfusion mismatch in lungs. As for the cellular mechanism of hypoxic pulmonary vasoconstriction (HPV), hypoxic inhibition of voltage-gated K+ channel (Kv) in PA smooth muscle cell (PASMC) has been suggested. In addition, our recent study showed that thromboxane A2 (TXA2) and hypoxia-activated nonselective cation channel (I(NSC)) is also essential for HPV. However, it is not well understood whether HPV is maintained in the animals exposed to ambient hypoxia for two days (2d-H). Specifically, the associated electrophysiological changes in PASMCs have not been studied. Here we investigate the effects of 2d-H on HPV in isolated ventilated/perfused lungs (V/P lungs) from rats. HPV was almost abolished without structural remodeling of PA in 2d-H rats, and the lost HPV was not recovered by Kv inhibitor, 4-aminopyridine. Patch clamp study showed that the hypoxic inhibition of Kv current in PASMC was similar between 2d-H and control. In contrast, hypoxia and TXA2-activated I(NSC) was not observed in PASMCs of 2d-H. From above results, it is suggested that the decreased I(NSC) might be the primary functional cause of HPV disappearance in the relatively early period (2 d) of hypoxia.

Buffering Contribution of Mitochondria to the Ca2+i Increase by Ca2+ Influx through Background Nonselective Cation Channels in Rabbit Aortic Endothelial Cells

To prove the buffering contribution of mitochondria to the increase of intracellular Ca2+ level ([Ca2+]i) via background nonselective cation channel (background NSCC), we examined whether inhibition of mitochondria by protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) affects endothelial Ca2+ entry and Ca2+ buffering in freshly isolated rabbit aortic endothelial cells (RAECs). The ratio of fluorescence by fura-2 AM (R340/380) was measured in RAECs. Biological state was checked by application of acetylcholine (ACh) and ACh (10microM) increased R340/380 by 1.1+/-0.15 (mean+/-S.E., n=6). When the external Na+ was totally replaced by NMDG+, R340/380 was increased by 1.19+/-0.17 in a reversible manner (n=27). NMDG+-induced [Ca2+]i increase was followed by oscillatory decay after [Ca2+]i reached the peak level. The increase of [Ca2+]i by NMDG+ was completely suppressed by replacement with Cs+. When 1microM CCCP was applied to bath solution, the ratio of [Ca2+]i was increased by 0.4+/-0.06 (n=31). When 1microM CCCP was used for pretreatment before application of NMDG+, oscillatory decay of [Ca2+]i by NMDG+ was significantly inhibited compared to the control (p < 0.05). In addition, NMDG+-induced increase of [Ca2+]i was highly enhanced by pretreatment with 2microM CCCP by 320+/-93.7%, compared to the control (mean+/-S.E., n=12). From these results, it is concluded that mitochondria might have buffering contribution to the [Ca2+]i increase through regulation of the background NSCC in RAECs.

Higher Expression of TRPM7 Channels in Murine Mature B Lymphocytes than Immature Cells

TRPM7, a cation channel protein permeable to various metal ions such as Mg2+, is ubiquitously expressed in variety of cells including lymphocytes. The activity of TRPM7 is tightly regulated by intracellular Mg2+, thus named Mg2+-inhibited cation (MIC) current, and its expression is known to be critical for the viability and proliferation of B lymphocytes. In this study, the level of MIC current was compared between immature (WEHI-231) and mature (Bal-17) B lymphocytes. In both cell types, an intracellular dialysis with Mg2+-free solution (140 mM CsCl) induced an outwardly-rectifying MIC current. The peak amplitude of MIC current and the permeability to divalent cation (Mn2+) were several fold higher in Bal-17 than WEHI-231. Also, the level of mRNAs for TRPM7, a molecular correspondence of the MIC channel, was significantly higher in Bal-17 cells. The amplitude of MIC was further increased, and the relation between current and voltage became linear under divalent cation-free conditions, demonstrating typical properties of the TRPM7. The stimulation of B cell receptors (BCR) by ligation with antibodies did not change the amplitude of MIC current. Also, increase of extracellular [Mg2+]c to enhance the Mg2+ influx did not affect the BCR ligation-induced death of WEHI-231 cells. Although the level of TRPM7 was not directly related with the cell death of immature B cells, the remarkable difference of TRPM7 might indicate a fundamental change in the permeability to divalent cations during the development of B cells.