Search

Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34− and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34− cells (p = 0.03), whereas mRNA of the mesenchymal marker Pdgfrα (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a Pdgfrα positive subpopulation within Epcam+/Sca-1+/CD34− epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34− cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,− cells in the biology of the murine lung. [ABSTRACT FROM AUTHOR]

Background: Two pertussis toxin sensitive Gi proteins, Gi2 and Gi3, are expressed in cardiomyocytes and upregulated in heart failure. It has been proposed that the highly homologous Gi isoforms are functionally distinct. To test for isoformspecific functions of Gi proteins, we examined their role in the regulation of cardiac L-type voltage-dependent calcium channels (L-VDCC). Methods: Ventricular tissues and isolated myocytes were obtained from mice with targeted deletion of either G&agr;i2 (Ga&agr;i2-/-) or G&agr;i3 (Ga&agr;i3-/-)). mRNA levels of Gai/o isoforms and L-VDCC subunits were quantified by real-time PCR. Gai and Cava1 protein levels as well as protein kinase B/Akt and extracellular signal-regulated kinases 1/2 (ERK1/2) phosphorylation levels were assessed by immunoblot analysis. L-VDCC function was assessed by whole-cell and single-channel current recordings. Results: In cardiac tissue from G&agr;i2-/- mice, G&agr;i3 mRNA and protein expression was upregulated to 187±21% and 567±59%, respectively. In G&agr;i3-/- mouse hearts, G&agr;i2 mRNA (127±5%) and protein (131±10%) levels were slightly enhanced. Interestingly, L-VDCC current density in cardiomyocytes from G&agr;i2-/- mice was lowered (-7.9±0.6 pA/pF, n = 11, p<0.05) compared to wild-type cells (-10.7±0.5 pA/pF, n = 22), whereas it was increased in myocytes from G&agr;i2-/- mice (-14.3±0.8 pA/pF, n = 14, p<0.05). Steady-state inactivation was shifted to negative potentials, and recovery kinetics slowed in the absence of G&agr;i2 (but not of G&agr;i3) and following treatment with pertussis toxin in G&agr;i3-/-. The pore forming Cav&agr;1 protein level was unchanged in all mouse models analyzed, similar to mRNA levels of Cav&agr;1 and Cav&bgr;2 subunits.Interestingly, at the cellular signalling level, phosphorylation assays revealed abolished carbachol-triggered activation of ERK1/2 in mice lacking G&agr;i2. Conclusion: Our data provide novel evidence for an isoform-specific modulation of L-VDCC by Gai proteins. In particular, loss of G&agr;i2 is reflected by alterations in channel kinetics and likely involves an impairment of the ERK1/2 signalling pathway. [ABSTRACT FROM AUTHOR]