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

Abstract

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.

Figure 1. Ca_v3.1^−/−;Ca_v3.2^−/− popliteal lymphatics are more sensitive to inhibition by NIF than WT lymphatics.

A) Response of a WT popliteal lymphatic vessel to increasing concentrations of NIF (applied cumulatively). Each contraction is a downward deflection (individual contractions cannot be resolved with this compressed time scale). Vertical lines are intentional artifacts created by blanking the light path to mark when a new concentration was added, followed by ~10 secs of mixing. Pressure was held constant at 2 cmH₂O. The cumulative DMSO concentration was < 0.4% and without effect alone. B) Response of a Ca_v3.1^−/−;Ca_v3.2^−/− popliteal lymphatic to the same NIF protocol. Contractions in the Ca_v3.1^−/−;Ca_v3.2^−/− vessel are completely inhibited at 100 nM NIF whereas the WT vessel requires at least 300 nM NIF to block contractions. C) Summary data for normalized AMP (normalized to the average AMP during the control period) as a function of NIF concentration. The curve for the Ca_v3.1^−/−;Ca_v3.2^−/− vessels is shifted to the left by ~1/2 log order, with two concentrations being significantly different. Summary data for FPF (D) and Frequency (E) as a function of NIF concentration. One concentration was significantly different for each parameter. F) Summary data for Normalized FREQ as a function of NIF concentration (normalized to the average FREQ during the control period). Two concentrations were significantly different and the curve for the Ca_v3.1^−/−;Ca_v3.2^−/− vessels was shifted to the left by ~1 log order. Statistical tests were two-way repeated measures ANOVAs with Tukey’s multiple comparison post-hoc tests (*, p<0.05). WT: N=5; n=9. Ca_v3 DKO: N=8; n=15.

Figure 2. Rationale for using PIN-induced hyperpolarization to enhance Ca_v3 current.

A) Theoretical depiction of window currents for NaV1, Ca_v3 and Ca_v1.2 channels relative to the resting Vm of a mouse LMC. B) Depiction of the effects of NIF and PIN on Vm relative to the window currents for the three types of voltage-gated cation channels. C). Experimental recording of Vm made with a sharp electrode in a mouse LMC during the application of 1 mM NIF followed by successive addition of 300 nM, 1 mM and 3 mM PIN. The vessel was treated with 2 mM wortmannin for 20 min prior to PIN application to blunt contractions (from ~50 to 5 mm) and reduce the chance of the electrode dislodging. NIF produced ~8 mV depolarization and blocks spontaneous APs. Successively higher concentrations of PIN reversed the depolarization into a net hyperpolarization. D) Summary data for the effects of NIF (1 mM) followed by 300 nM, 1 mM and 3 mM PIN. Glibenclamide (GLIB, 3 mM) reversed the effects of PIN. Statistical significance was determined using a mixed effects ANOVA with Tukey’s post-hoc test (*, p<0.05; **, p<0.05; ***, p<0.005; ****, p<0.005). N=12; n=12.

Figure 3. Protocol for determining the effects of PIN amplitude of EFS-evoked contractions during inhibition of NaV and Ca_v1.2 channels.

A) Control measurements of spontaneous contraction amplitude were made over at least a 2-min period prior to the addition of any drug. TTX (1 mM) was applied for 5–8 min followed by NIF (1 mM) for 5–6 min prior to the addition of the three successive concentrations of PIN (2–3 min each). Three EFS pulses (0.1–0.3 ms, 80–90 V) were delivered ~30–60 sec apart during each period of drug application. B) Pressure and diameter recordings from a representative WT popliteal lymphatic to illustrate the results of the protocol. Pin and Pout are the respective inflow and outflow pressures, which were set to equal levels and held constant throughout the protocol. The bath was exchanged for Ca²⁺-free Krebs at the end of the protocol and after 30 min the maximum passive diameter was obtained for calculation of normalized diameter.

Figure 4. Representative recordings of EFS-evoked contractions in the three genotypes to the Pharmacological treatments.

A-C) WT vessels. D-E) Ca_v1.2 smKO vessels (NIF treatments in D and E were unnecessary). F-H) Ca_v3 DKO vessels. In each case the concentrations were TTX (1 mM), NIF (1 mM), PIN (3 mM). All EFS pulses were 0.2 msec at 150 V except in A, which was 0.1 msec.

Figure 5. Summary data for the amplitudes of spontaneous and EFS-evoked contractions in vessels of the three genotypes during NIF and PIN treatment.

A) For WT vessels there were no significant differences in the amplitudes of spontaneous and EFS-evoked contractions, but the differences between both of those and the amplitudes of EFS-evoked contractions in NIF and NIF + PIN were significant (****, only the comparisons to the NIF + largest AMP of the combined PIN groups are marked). PIN significantly enhanced the AMP of EFS-evoked pulses compared to NIF alone (###). B) Spontaneous contraction amplitudes were <3 mm in Ca_v1.2 smKO vessels and EFS-evoked contractions were even smaller; the difference between the average AMP was not significant. 1 mM and 3 mM PIN significantly enhanced the AMP of EFS-evoked contractions (##, only marked for the largest PIN AMP). C) For Ca_v3 DKO vessels spontaneous contractions occurred and there were no significant differences in the amplitudes of spontaneous and EFS-evoked contractions. However, the differences between the amplitudes of both spontaneous and EFS-evoked contractions and the amplitudes of EFS-evoked contractions in NIF and NIF + PIN were significant (****, only the comparison between the NIF + largest AMP with PIN is marked). In the presence of NIF, EFS-evoked contractions were <3 mm in amplitude and were not significantly enhanced by PIN. ****; p<0.001, one-way ANOVA. ###; p<0.001, Wilcoxon paired signed rank test. ##; p<0.01, Wilcoxon paired signed rank test. Ns = not significant at p<0.05, Wilcoxon paired signed rank test. WT: N=7; n=12–14. Ca_v1.2 smKO: N=4; n=9. Ca_v3 DKO: N=5; n=8.