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(v)3) channels (Ca(v)3.1, Ca(v)3.2) that are functionally expressed in murine lymphatic muscle cells; however, contractile tests of lymphatic vessels from single and double Ca(v)3 knock-out (DKO) mice, exhibited nearly identical parameters of spontaneous twitch contractions as wild-type (WT) vessels, suggesting that Ca(v)3 channels play no significant role. Here, we considered the possibility that the contribution of Ca(v)3 channels might be too subtle to detect in standard contraction analyses. We compared the sensitivity of lymphatic vessels from WT and Ca(v)3 DKO mice to the L-type calcium channel (Ca(v)1.2) inhibitor nifedipine and found that the latter vessels were significantly more sensitive to inhibition, suggesting that the contribution of Ca(v)3 channels might normally be masked by Ca(v)1.2 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(v)3 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(v)1.2 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(v)3 DKO vessels. Our results point to a subtle contribution of Ca(v)3 channels to lymphatic contractions that can be unmasked in the absence of Ca(v)1.2 channel activity and when the resting Vm is more hyperpolarized than normal.