Abstract
The spontaneous contractions of collecting lymphatic vessels provide an essential propulsive force to return lymph centrally. These contractions are driven by an intrinsic electrical pacemaker, working through an unknown underlying ionic mechanism that becomes compromised in some forms of lymphedema. In previous studies, T-type voltage-gated Ca2+ channels (VGCCs) were implicated in this pacemaking mechanism, based on the effects of the reputedly selective T-type VGCC inhibitors mibefradil and Ni2+. Our goal was to test this idea in a more definitive way using genetic knock out mice. First, we demonstrated through both PCR and immunostaining that mouse lymphatic muscle cells expressed Cav3.1 and Cav3.2 and produced functional T-type VGCC currents when patch clamped. We then employed genetic deletion strategies to selectively test the roles of each T-type VGCC isoform in the regulation of lymphatic pacemaking. Surprisingly, global deletion of either, or both, isoform(s) was without significant effect on either the frequency, amplitude, or fractional pump flow of lymphatic collectors from two different regions of the mouse, studied ex vivo. Further, both WT and Cav3.1−/−; 3.2−/− double knock-out lymphatic vessels responded similarly to mibefradil and Ni2+, which substantially reduced contraction amplitudes and slightly increased frequencies at almost all pressures in both strains: a pattern consistent with inhibition of L-type rather than T-type VGCCs. Neither T-type VGCC isoform was required for ACh-induced inhibition of contraction, a mechanism by which those channels in smooth muscle are thought to be targets of endothelium-derived nitric oxide. Sharp intracellular electrode measurements in lymphatic smooth muscle revealed only subtle, but not significant, differences in the resting membrane potential and action potential characteristics between vessels from wild-type and Cav3.1−/−; 3.2−/− double knock-out mice. In contrast, smooth-muscle specific deletion of the L-type VGCC, Cav1.2, completely abolished all lymphatic spontaneous contractions. Collectively our results suggest that, although T-type VGCCs are expressed in mouse lymphatic smooth muscle, they do not play a significant role in modulating the frequency of the ionic pacemaker or the amplitude of spontaneous contractions. We conclude that the effects of mibefradil and Ni2+ in other lymphatic preparations are largely or completely explained by off-target effects on L-type VGCCs, which are essential for controlling both the frequency and strength of spontaneous contractions.
Highlights
Two studies have implicated T-type VGCCs as contributors to pacemaking in lymphatic vessels
Prior to performing experiments on mouse lymphatic vessels, we sought to confirm the expression of Cav3.1 and Cav3.2 in rat mesenteric lymphatic smooth muscle cells (LMCs) and the effects of the T-type VGCC inhibitors mibefradil and Ni2+ on rat mesenteric lymphatics as reported by Lee et al.[35]
With pressure maintained at that level, mibefradil was added to the bath in cumulative concentrations while assessing its effects on contraction amplitude and frequency for 2 min at each concentration
Summary
Two studies have implicated T-type VGCCs as contributors to pacemaking in lymphatic vessels. To address the roles of VGCCs in LMCs in a way that does not rely on poorly selective inhibitors, we used genetic approaches to delete specific L- or T-type VGCC isoforms from mouse LMCs. Using popliteal afferent lymphatics and inguinal-axillary lymphatics (IALs) as models of robustly contracting peripheral lymphatic vessels, we first identified which Cav[3] and Cav1.2 isoforms were expressed, used patch clamp methods to verify the presence of functional Cav[3] channels based on their kinetics and voltage dependency, and studied the contractile function and characteristics of the LMC action potential in lymphatic vessels from mice with global- or smooth muscle-specific deletions of those isoforms. Our results lead to the surprising conclusion that T-type VGCCs do not play any significant role in controlling the pacemaking frequency or contributing to the amplitude of lymphatic spontaneous contractions, while L-type VGCCs are essential for both
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