Electric field stimulation unmasks a subtle role for T-type calcium channels in regulating lymphatic contraction

We previously identified two isoforms of T-type, voltage-gated calcium (Ca_v3) channels (Ca_v3.1, Ca_v3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Ca_v3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Ca_v3 channels play no significant role. Here, we considered the possibility that the contribution of Ca_v3 channels might be too subtle to detect in standard contraction analyses. We compared the sensitivity of lymphatic vessels from WT and Ca_v3 DKO mice to the L-type calcium channel (Ca_v1.2) inhibitor nifedipine and found that the latter vessels were significantly more sensitive to inhibition, suggesting that the contribution of Ca_v3 channels might normally be masked by Ca_v1.2 channel activity. We hypothesized that shifting the resting membrane potential (Vm) of lymphatic muscle to a more negative voltage might enhance the contribution of Ca_v3 channels. Because even slight hyperpolarization is known to completely silence spontaneous contractions, we devised a method to evoke nerve-independent, twitch contractions from mouse lymphatic vessels using single, short pulses of electric field stimulation (EFS). TTX was present throughout to block the potential contributions of voltage-gated Na⁺ channels in perivascular nerves and lymphatic muscle. In WT vessels, EFS evoked single contractions that were comparable in amplitude and degree of entrainment to those occurring spontaneously. When Ca_v1.2 channels were blocked or deleted, only small residual EFS-evoked contractions (~ 5% of normal amplitude) were present. These residual, EFS-evoked contractions were enhanced (to 10-15%) by the K_ATP channel activator pinacidil (PIN) but were absent in Ca_v3 DKO vessels. Our results point to a subtle contribution of Ca_v3 channels to lymphatic contractions that can be unmasked in the absence of Ca_v1.2 channel activity and when the resting Vm is more hyperpolarized than normal.