Abstract
Active lymph pumping relies on the spontaneous contractions of collecting lymphatic vessels, with frequencies that 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 capillaries. We investigated the mechanistic basis of pressure-induced chronotropy using ex vivo contraction assays of mouse popliteal collecting vessels, in which contraction frequency increased >10-fold with a 5 cmH(2)O pressure change. The contractile, electrophysiological and transcriptional similarities between lymphatic muscle and arterial smooth muscle led us to hypothesize that pressure-dependent chronotropy shares a parallel signaling process to pressure-induce arterial depolarization/constriction. Thus, we investigated 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 GPCRs that would generate second messengers to activate those channels. We combined contraction assays with scRNAseq analysis of the respective targets and made maximum use 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 TRP and other mechanosensitive channels implicated in myogenic constriction, as well as channels implicated in ionic pacemaking of other tissues, and instead support a scheme whereby mechano-activation of GNAQ/GNA11-coupled GPCRs generates IP(3), which induces SR Ca(2+) release through IP(3)R1 and drives depolarization through the activation of ANO1 Cl(-) channels.