Calmodulin (CaM) has been shown to modulate different ion channels, including voltage-gated sodium channels (NaChs). Using the yeast two-hybrid assay, we found an interaction between CaM and the C-terminal domains of adult skeletal (Na V 1.4) and cardiac (Na V 1.5) muscle NaChs. Effects of CaM were studied using sodium channels transiently expressed in CHO cells. Wild type CaM (CaM WT ) caused a hyperpolarizing shift in the voltage dependence of activation and inactivation for Na V 1.4 and activation for Na V 1.5. Intracellular application of CaM caused hyperpolarizing shifts equivalent to those seen with CaM WT coexpression with Na V 1.4. Elevated Ca 2+ and CaM-binding peptides caused depolarizing shifts in the inactivation curves seen with CaM WT coexpression with Na V 1.4. KN93, a CaM-kinase II inhibitor, had no effect on Na V 1.4, suggesting that CaM acts directly on Na V 1.4 and not through activation of CaM-kinase II. Coexpression of hemi-mutant CaMs showed that an intact N-terminal lobe of CaM is required for effects of CaM upon Na V 1.4. Mutations in the sodium channel IQ domain disrupted the effects of CaM on Na V 1.4: the I1727E mutation completely blocked all calmodulin effects, while the L1736R mutation disrupted the effects of Ca 2+ -calmodulin on inactivation. Chimeric channels of Na V 1.4 and Na V 1.5 also indicated that the C-terminal domain is largely responsible for CaM effects on inactivation. CaM had little effect on Na V 1.4 expressed in HEK cells, possibly due to large differences in the endogenous expression of β-subunits between CHO and HEK cells. These results in heterologous cells suggest that Ca 2+ released during muscle contraction rapidly modulates NaCh availability via CaM.
Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45(pos)/Flk-1(pos) and CD45(neg)/Flk-1(pos) bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.
Bone morphogenetic proteins (BMPs) have been implicated in the pathogenesis of familial pulmonary arterial hypertension. The type 2 receptor (BMPR2) is required for recognition of all BMPs. Transgenic mice with a smooth muscle cell-targeted mutation in this receptor (SM22-tet-BMPR2 delx4ϩ ) developed increased pulmonary artery pressure, associated with a modest increase in arterial muscularization, after 8 wk of transgene activation (West ). In the present study, we show that these transgenic mice developed increased right ventricular pressures after only 1 wk of transgene activation, without significant remodeling of the vasculature. We then tested the hypothesis that the increased pulmonary artery pressure due to loss of BMPR2 signaling was mediated by reduced K V channel expression. There was decreased expression of K V1.1, KV1.5, and KV4.3 mRNA isolated from whole lung. Western blot confirmed decreased K V1.5 protein in these lungs. Human pulmonary artery smooth muscle cells (PASMC) treated with recombinant BMP2 had increased K V1.5 protein and macroscopic KV current density, which was blocked by anti-KV1.5 antibody. In vivo, nifedipine, a selective L-type Ca 2ϩ channel blocker, reduced RV systolic pressure in these dominant-negative BMPR2 mice to levels seen in control animals. This suggests that activation of L-type Ca 2ϩ channels caused by reduced KV1.5 mediates increased pulmonary artery pressure in these animals. These studies suggest that BMP regulates K V channel expression and that loss of this signaling pathway in PASMC through a mutation in BMPR2 is sufficient to cause pulmonary artery vasoconstriction. pulmonary arterial hypertension; voltage-gated potassium channel 1.5; bone morphogenetic protein receptor type 2; vascular tone A POTENTIAL ROLE for bone morphogenetic protein (BMP) signaling in the pathogenesis of vascular disease was suggested by studies of hereditary pulmonary arterial hypertension (PAH), a disorder characterized by the pathological development of increased pressure and structural remodeling of the pulmonary circulation. Mutations in the BMP type 2 receptor (BMPR2) gene were found to be responsible for hereditary PAH (10, 17). Subsequently, sporadic cases of PAH were also found to be associated with mutations in BMPR2 (37).Transgenic mice have demonstrated a critical role for BMP signaling in development. BMPR2(Ϫ/Ϫ) mice die early in development, before gastrulation. BMPR2(ϩ/Ϫ) mice develop normally and were originally thought to have no cardiovascular phenotype (4). However, a recent report in BMPR2(ϩ/Ϫ) mice suggested that they may have modest pulmonary hypertension, though that finding was not verified by a second group (18). Our laboratory constructed a conditional transgenic mouse expressing a dominant-negative BMPR2 (dnBMPR2) selectively in smooth muscle cells (SM22-tet-BMPR2 delx4ϩ mice). When the mutation was activated for 2 mo immediately after birth, the mice developed increased pulmonary artery pressure associated with a modest increase in arterial muscularization (39)....
Pulmonary hypertension (PH) is characterized by sustained vasoconstriction, with subsequent extracellular matrix (ECM) production and smooth muscle cell (SMC) proliferation. Changes in the ECM can modulate vasoreactivity and SMC contraction. Galectin-1 (Gal-1) is a hypoxia-inducible beta-galactoside-binding lectin produced by vascular, interstitial, epithelial, and immune cells. Gal-1 regulates SMC differentiation, proliferation, and apoptosis via interactions with the ECM, as well as immune system function, and, therefore, likely plays a role in the pathogenesis of PH. We investigated the effects of Gal-1 during hypoxic PH by quantifying 1) Gal-1 expression in response to hypoxia in vitro and in vivo and 2) the effect of Gal-1 gene deletion on the magnitude of the PH response to chronic hypoxia in vivo. By constructing and screening a subtractive library, we found that acute hypoxia increases expression of Gal-1 mRNA in isolated pulmonary mesenchymal cells. In wild-type (WT) mice, Gal-1 immunoreactivity increased after 6 wk of hypoxia. Increased expression of Gal-1 protein was confirmed by quantitative Western analysis. Gal-1 knockout (Gal-1(-/-)) mice showed a decreased PH response, as measured by right ventricular pressure and the ratio of right ventricular to left ventricular + septum wet weight compared with their WT counterparts. However, the number and degree of muscularized vessels increased similarly in WT and Gal-1(-/-) mice. In response to chronic hypoxia, the decrease in factor 8-positive microvessel density was similar in both groups. Vasoreactivity of WT and Gal-1(-/-) mice was tested in vivo and with use of isolated perfused lungs exposed to acute hypoxia. Acute hypoxia caused a significant increase in RV pressure in wild-type and Gal-1(-/-) mice; however, the response of the Gal-1(-/-) mice was greater. These results suggest that Gal-1 influences the contractile response to hypoxia and subsequent remodeling during hypoxia-induced PH, which influences disease progression.
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