Roles of G-protein coupled receptors and mechanosensitive ion channels in pressure-induced chronotropy of lymphatic vessels

Abstract

Active lymph pumping relies on the spontaneous contractions of collecting lymphatic vessels, whose contraction frequencies are exquisitely sensitive to changes in intraluminal pressure. This homeostatic and mechanosensitive mechanism, termed pressure-induced lymphatic chronotropy, enables lymph transport to be matched to the filling state of the lymphatic capillary network. The mechanistic basis of pressure-induced chronotropy was investigated using ex vivo contraction assays of mouse popliteal collecting vessels, in which contraction frequency increases >10-fold with pressure changed from 0.5 to 5 cmH2O. The contractile, electrophysiological and transcriptional similarities between lymphatic muscle cells (LMCs) and arterial smooth muscle led us to hypothesize that pressure-dependent chronotropy shares a parallel signaling process with pressure-induced arterial depolarization/constriction. Thus, we probed two major mechanisms: 1) pressure-induced activation of mechanosensitive cation channels, including TRPC6, TRPM4, PKD1/2, TRPV2 and ENaC, and 2) mechano-activation of GNAQ/GNA11-coupled G-protein receptors (GPCRs) that would generate second messengers to activate those channels. Contraction assays were combined with scRNAseq analysis of the respective targets, with maximum use made of transgenic mice to avoid non-specific effects of pharmacological inhibitors, particularly those used to block TRP channels. Our findings rule out significant roles for the above TRP channels and other putative mechanosensitive channels implicated in arterial myogenic constriction, as well as channels implicated in ionic pacemaking of other tissues. In contrast, smooth-muscle specific knock out or inhibition of ANO1 or IP3R1 significantly blunted the effect of pressure on frequency. Pressure-induced chronotropy was suppressed by [~]70-90% at all pressures in GNAQ/GNA11 double knockout vessels, but with responsiveness partially maintained at pressures above 5 cmH2O. Pressure-induced chronotropy was also suppressed after acute Gq/11 inhibition with YM254890, but was normal in vessels from GNA12/GNA13 double knock out mice. These results support a scheme whereby mechano-activation of one or more GNAQ/GNA11-coupled GPCRs generates IP3, which induces SR Ca2+ release through IP3R1 and drives depolarization through the activation of ANO1 Cl- channels. The major GPCRs expressed in LMCs were subsequently identified and ranked by scRNAseq analysis but knock out or pharmacological inhibition of each of the top 7 candidates failed to significantly affect pressure-induced chronotropy. Our results strongly implicate one or more GNAQ/GNA11-coupled GPCRs in mediating this homeostatic process; however, the specific mechanosensitive GPCRs remain to be identified.