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dc.contributor.advisorDavis, Michaeleng
dc.contributor.authorTo, Kim Hung Thieneng
dc.date.issued2017eng
dc.date.submitted2017 Springeng
dc.description.abstractLymphatic smooth muscle (LSM) contracts spontaneously, actively returning interstitial fluid through a network of lymphatic capillaries and collecting lymphatic vessels to the great veins. Dysfunctional lymphatic contractions can impair lymph transport in lymphatic-related diseases such as lymphedema. Understanding the pacemaking mechanism of LSM that underlies active lymph transport is essential for therapeutic targeting of lymphedema. Based on experiments using pharmacological inhibitors, current literature posits that T-type voltage-gated Ca2+ channels (T-channels) play a role in controlling the pacing of lymphatic contractions, i.e., the contraction frequency, while Ltype voltage-gated Ca2+ channels (L-channels) play a role in controlling the strength of lymphatic contractions, i.e., the contraction amplitude. However, non-specific effects of currently available T-channel inhibitors, especially on L-channels, can confound the understanding of T-channel role in lymphatic pacemaking. Therefore, using transgenic mouse models as an alternative approach to test the role of T-channels, I hypothesized that genetic deletion of T-type Ca2+ channels would decrease the frequency of lymphatic contractions but not the amplitude. First, I tested for the presence of T-channels in lymphatic vessels from both rat and mouse, and then more specifically in isolated single mouse LSM cells; second, I tested the effects of commonly-used T-channel inhibitors on lymphatic pacemaking and/or contraction in both rat and mouse vessels; and finally, I investigated the effect of genetic deletion of specific T-channel isoforms in mice on lymphatic pacemaking and contraction strength. First, RT-PCR and immunostaining were performed on whole lymphatic vessels to test for the expression of T-channels at mRNA and protein levels. Rat mesenteric lymphatics, mouse popliteal lymphatic vessels (PLs) and mouse inguinal-axillary lymphatic vessels (IALs) showed the mRNA expression of Cav3.1 and 3.2, two of the three isoforms of T-channels, along with Cav1.2, the isoform of the L-channel prevalent in cardiac muscle and blood vessels. Likewise, in LSM cells isolated from mouse PLs and IALs, RT-PCR revealed the expression of Cav3.1 and 3.2. In mouse IALs, immunostaining consistently revealed the protein expression of T-channel isoforms Cav3.1 and 3.2 along with L-channel isoform Cav1.2 colocalized with the smooth-muscle a-actin (i.e., in LSM cells). Moreover, patch-clamp recordings in single LSM cells isolated from rat mesenteric, mouse PLs and IALs showed functional evidence of current through voltage-gated Ca2+ channels that was blocked by 1[mu]M nifedipine, an L-channel inhibitor, along with a persistent nifedipine-insensitive current that had fast kinetics and was blocked by 1mM Ni2+, a frequently used T-channel inhibitor. Second, pharmacological inhibitors were tested on isolated, cannulated and pressurized ex vivo lymphatic vessels from rat and mouse. Consistent with the findings of Lee et al. (2014) on rat mesenteric lymphatics, mibefradil, another conventional T-channel inhibitor, inhibited the contraction frequency (IC50=66nM) at a lower dose than that required to inhibit contraction amplitude (IC50=423nM). However, in contrast to their findings, treatment of rat mesenteric lymphatics with Ni2+ inhibited both amplitude and frequency at similar doses (IC50=248µM and 279[mu]M, respectively). In wild-type (WT) mouse IALs and PLs, increasing doses of Ni2+ progressively reduced contraction amplitude (IC50=66[mu]M and 110[mu]M, respectively), while leaving the frequency unchanged until the contractions were completely inhibited. In WT PLs, TTA-A2, a more recently developed T-channel inhibitor, had only a modest effect on contraction amplitude (IC50=1.3[mu]M) without changing the contraction frequency. Similarly, treatment with nifedipine, a specific L-channel inhibitor, gradually attenuated contraction amplitude (IC50=43.3nM), suggesting that the effect on amplitude of T-channel inhibitors Ni2+ and TTA-A2 could be due to off-target effects on L-channels. Having established that pharmacologic inhibition of T-channels in this context is unreliable, I turned to genetic methods allowing deletion of specific T and L-channel isoforms. Surprisingly, smooth muscle-specific deletion of Cav1.2 (L-channels) rendered PLs and IALs quiescent without spontaneous lymphatic contractions, suggesting their potential contribution to both lymphatic frequency and contraction strength; no residual contractions were mediated by T-channels. In Cav3.1-null mice and Cav3.2-null mice, IALs exhibited no significant differences in functional contractile parameters (including frequency and amplitude) compared to WT vessels over a wide range of pressures. Likewise, PLs from Cav3.1-/- mice exhibited no significant defects in the contractile response to pressure, to the L-channel inhibitor nifedipine, or even to the endothelialdependent inhibitor acetylcholine. These findings conflict with the currently established view that T-channels regulate the frequency of lymphatic pacemaking and that L-channels contribute only to the contraction strength. In summary, I confirmed the functional expression of T-channels in both rat and mouse LSM, but selective genetic deletion of either Cav3.1 or Cav3.2 T-channel isoforms did not produce a measurable functional defect in lymphatic vessel pacemaking or contraction. My findings conflict with the current established view that T-channels control lymphatic pacemaking and L-channels determine lymphatic contraction strength; a definitive role for T-channels in LSM function remains unknown.eng
dc.description.bibrefIncludes biblographical referenceseng
dc.format.extentxix, 183 pages : illustrationeng
dc.identifier.urihttps://hdl.handle.net/10355/62263
dc.languageEnglisheng
dc.publisherUniversity of Missouri--Columbiaeng
dc.relation.ispartofcollectionUniversity of Missouri--Columbia. Graduate School. Theses and Dissertationseng
dc.rightsOpenAccesseng
dc.rights.licenseThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.eng
dc.subject.FASTLymphaticseng
dc.subject.FASTCalcium channelseng
dc.subject.FASTCardiac pacemakerseng
dc.titleRole of T-type Ca[2+] channels in lymphatic pacemakingeng
dc.typeThesiseng
thesis.degree.disciplinePharmacology (MU)eng
thesis.degree.grantorUniversity of Missouri--Columbiaeng
thesis.degree.levelDoctoraleng
thesis.degree.namePh. D.eng


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