In functional arterial studies using wire myography, the determination of a vessel’s standardized normalization factor (factor k) is an essential step to ensure optimal contraction and relaxation by the arteries when stimulated with their respective vasoactive agents and to obtain reproducible results. The optimal factor k for several arteries have been determined, however, the optimal initial tension and factor k for the arteries involved in erection remains unknown. Hence, in the present study we set out to determine the optimal factor k for the internal iliac artery, proximal and distal internal pudendal artery (IPA), and dorsal penile artery. After isolating, harvesting, and mounting the arteries from male Sprague-Dawley rats on a multi wire myograph, we tested arterial responsivity to high K+-stimulation when the factor k was set at 0.7, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, and 1.2 to determine the factor k setting that results in the greatest K+-induced active force production for each vessel type. The data showed the optimal factor k is 0.90-0.95 for the dorsal penile, distal internal pudendal and internal iliac arteries while it is 0.85-0.90 for proximal internal pudendal artery. These optimal values corresponded to initial passive tension settings of 1.10±0.16 - 1.46±0.23, 1.28±0.20 - 1.69±0.34, 1.03±0.27 - 1.33±0.31, and 1.33±0.31 - 1.77±0.43 mN/mm for the dorsal penile, distal IP, proximal IP, and internal iliac arteries, respectively.
Exposure to hypoxia, due to high altitude or chronic lung disease, leads to structural changes in the pulmonary vascular wall, including hyperplasia and migration of pulmonary arterial smooth muscle cells (PASMCs). Previous studies showed that hypoxia upregulates the expression of Na+/H+ exchanger isoform 1 (NHE1) and that inhibition or loss of NHE1 prevents hypoxia-induced PASMC migration and proliferation. The exact mechanism by which NHE1 controls PASMC function has not been fully delineated. In fibroblasts, NHE1 has been shown to act as a membrane anchor for actin filaments, via binding of the adaptor protein, ezrin. Thus, in this study, we tested the role of ezrin and NHE1/actin interactions in controlling PASMC function. Using rat PASMCs exposed to in vitro hypoxia (4% O2, 24 h) we found that hypoxic exposure increased phosphorylation (activation) of ezrin, and promoted interactions between NHE1, phosphorylated ezrin and smooth muscle specific α-actin (SMA) as measured via immunoprecipitation and co-localization. Overexpression of wild-type human NHE1 in the absence of hypoxia was sufficient to induce PASMC migration and proliferation, whereas inhibiting ezrin phosphorylation with NSC668394 suppressed NHE1/SMA co-localization and migration in hypoxic PASMCs. Finally, overexpressing a version of human NHE1 in which amino acids were mutated to prevent NHE1/ezrin/SMA interactions was unable to increase PASMC migration and proliferation despite exhibiting normal Na+/H+ exchange activity. From these results, we conclude that hypoxic exposure increases ezrin phosphorylation in PASMCs, leading to enhanced ezrin/NHE1/SMA interaction. We further speculate that these interactions promote anchoring of the actin cytoskeleton to the membrane to facilitate the changes in cell movement and shape required for migration and proliferation.
Pulmonary hypertension (PH) is a severe condition defined by mean pulmonary artery pressure >20 mmHg. PH pathogenesis involves vascular contraction and remodeling characterized by increased PASMC migration and proliferation. Na+/H+ exchanger isoform 1 (NHE1) is a membrane protein that regulates intracellular pH (pHi) and cell volume via export of H+ and import of Na+. This protein also has a cytosolic tail region shown to be involved in non-canonical functions such as cytoskeletal interactions via protein-protein interactions. Of particular interest, the tail region contains a binding site for ERM (ezrin/radixin/moesin) proteins. In other cell types, this binding site regulates actin filament organization, cell shape and migration by interacting with ezrin.We previously found NHE activity, and in some cases NHE1 expression, is increased in PASMCs from PH patients and animal models and was associated with enhanced proliferation and migration. Additionally, NHE inhibition with ethyl isopropyl amiloride reduced migration and proliferation in PASMCs from PH models; however, it is unclear if dysregulation of pHi or other functions of NHE1 are driving these abnormalities. In this study, we wanted to elucidate the mechanism by which NHE1 promotes increased PASMC migration and proliferation. We hypothesized dysregulation of pHi by NHE1 increases proliferation, whereas the interaction between NHE1 and ezrin mediates increased migration.PASMCs were isolated from distal pulmonary arteries of male Wistar rats. Cells were infected with adenovirus containing green fluorescent protein (AdGFP), wild-type NHE1 (AdNHE1-WT) or NHE1 mutants. To analyze the role of pHi regulation, a mutant containing a single amino acid substitution at position 262 was used to prevent ion translocation activity (AdNHE1-I). To analyze the role of protein-protein interactions, we generated a mutant in which a cluster of lysines and arginines in the ezrin binding region was replaced by alanines (AdNHE1-E). To confirm correct protein localization and efficacy of Na+/H+ translocation of the overexpressed proteins, NHE activity was assessed via Na+-dependent recovery following an ammonium pulse. Protein overexpression was confirmed via immunoblotting.Finally, cells underwent experiments to analyze migration (transwell assay) and proliferation (BrdU incorporation). All measurements were made in a blinded fashion.Enhanced expression of wildtype NHE1 increased PASMC migration and proliferation. Expression of the translocation deficient mutant (AdNHE1-I) also increased migration; however, proliferation was unchanged compared to AdGFP. Interestingly, AdNHE1-E (ezrin binding mutant) was unable to increase migration or proliferation. Our results suggest the protein-protein interaction between NHE1 and ezrin is essential for NHE1 to increase PASMC migration and proliferation. In contrast, pH regulation by NHE1 contributes to increased PASMC proliferation, but increased migration is pH-independent. R01HL073859 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
In functional arterial studies using wire myography, the determination of a vessel’s standardized normalization factor (factor k) is an essential step to ensure optimal contraction and relaxation by the arteries when stimulated with their respective vasoactive agents and to obtain reproducible results. The optimal factor k for several arteries have been determined; however, the optimal initial tension and factor k for the arteries involved in erection remains unknown. Hence, in the present study we set out to determine the optimal factor k for the internal iliac artery, proximal and distal internal pudendal artery (IPA), and dorsal penile artery. After isolating, harvesting, and mounting the arteries from male Sprague-Dawley rats on a multi wire myograph, we tested arterial responsivity to high K+-stimulation when the factor k was set at 0.7, 0.8, 0.85, 0.9, 0.95, 1.0, 1.1, and 1.2 to determine the factor k setting that results in the greatest K+-induced active force production for each vessel type. The data showed the optimal factor k is 0.90-0.95 for the dorsal penile, distal internal pudendal and internal iliac arteries while it is 0.85-0.90 for proximal internal pudendal artery. These optimal values corresponded to initial passive tension settings of 1.10±0.16 - 1.46±0.23, 1.28±0.20 - 1.69±0.34, 1.03±0.27 - 1.33±0.31, and 1.33±0.31 - 1.77±0.43 mN/mm for the dorsal penile, distal IP, proximal IP, and internal iliac arteries, respectively.
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