Background: Phenylalanine levels are associated with pulmonary hypertension in metabolic profiling clinical studies. However, the pathophysiologic role of phenylalanine on pulmonary circulation is still unclear. We experimentally addressed the direct impact of phenylalanine on pulmonary circulation in rats and explored the underlying molecular pathway. Methods and results: Phenylalanine was injected intraperitoneally into Sprague-Dawley rats (400 mg/100g body weight) as a single dose or daily in a chronic manner for 2, 3 and 4 weeks. Chronic injection of phenylalanine induced pulmonary hypertension with time-dependent severity evidenced by elevated pulmonary artery pressure and pulmonary vascular resistance, as well as pulmonary artery and right ventricular hypertrophy. Using tandem mass spectrometry analysis, we found a quick 2-fold increase in blood level of phenylalanine 2 hours following injection. This increase led to a significant accumulation of phenylalanine in lung after 4 hours which remained sustained at up to 3-fold increase after 4 weeks. In addition, cellular thermal shift assay with lung tissues from phenylalanine-injected rats reveals the binding of phenylalanine to the calcium-sensing receptor (CaSR). In vitro experiments with cultured pulmonary arterial smooth muscle cells showed that phenylalanine activated CaSR as indicated by the increase in intracellular calcium content, which was attenuated or diminished by the inhibition or knockdown of CaSR. Finally, the global knockout or lung-specific knockdown of CaSR significantly attenuated phenylalanine-induced pulmonary hypertension. Conclusions: Chronic phenylalanine injection induces pulmonary hypertension through binding to CaSR and its subsequent activation.

The effect of calcium sensing receptor (CaSR) on tumor cell proliferation has been studied in several human cancers, and great discrepancies were found in different tumors. However, the role of CaSR in lung adenocarcinomas (LUADs) is not clear. Therefore, we investigated the function of CaSR on regulating the growth of human LUAD and its possible mechanism. The expression of CaSR protein and its relationship with pathological parameters were examined in paraffin sections from 51 LUAD patients, by immunohistochemistry. The results showed that CasR expression was negatively correlated with the Ki-67 index as well as the grade of malignancy in LUAD. Further, CaSR demonstrated an in vitro inhibitory effect on the proliferation of human LUAD A549 cells by regulating CaSR activity with agonist cinacalcet, antagonist NPS2143, or shRNA-CaSR transfection. Tumor xenograft models also verified the in vivo proliferation-inhibiting role of CaSR by subcutaneous injecting A549 cells into nude mice with or without changes of CaSR activity. Molecularly, Western blotting showed that CaSR positively regulated the activity of glycogen synthase kinase 3β (GSK3β), followed by the downregulation of Cyclin D1. We used the dominant negative mutant and the constitutively active mutant plasmid of GSK3β to alter GSK3β activity. Our functional experiments showed that the proliferation-inhibition of CaSR was suppressed by the inactivation of GSK3β and enhanced by the activation of GSK3β. These results suggested that CaSR played a proliferation-inhibiting role in LUAD, at least partially by regulating the GSK3β/Cyclin D1 pathway.