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Human lymphatic vessels are myogenically active and respond to sympathetic stimulation. The role of various cations in this behavior has recently been investigated, but whether the anion Cl− is essential is unclear. With ethical approval and informed consent, human thoracic duct and mesenteric lymphatic vessels were obtained from surgical patients. Spontaneous or norepinephrine-induced isometric force production from isolated vessels was measured by wire myography; the transmembrane Cl− gradient and Cl− channels were investigated by substitution of extracellular Cl− with the impermeant anion aspartate and inhibition of Cl− transport and channels with the clinical diuretics furosemide and bendroflumethiazide as well as DIDS and 5-nitro-2-(3-phenylpropylamino)benzoic acid. The molecular expression of Ca2+-activated Cl− channels was investigated by RT-PCR, and proteins were localized using immunoreactivity. Spontaneous and norepinephrine-induced contractility in human lymphatic vessels was highly abrogated after Cl− substitution with aspartate. About 100–300 µM DIDS or 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibited spontaneous contractile behavior. Norepinephrine-stimulated tone was furthermore markedly abrogated by 200 µM DIDS. Furosemide lowered only spontaneous constrictions, whereas bendroflumethiazide had nonspecific inhibitory effects. Consistent expression of transmembrane member 16A [TMEM16A (anoctamin-1)] was found in both the thoracic duct and mesenteric lymphatic vessels, and immunoreactivity with different antibodies localized TMEM16A to lymphatic smooth muscle cells and interstitial cells. The significant change in contractile function observed with inhibitors and anion substitution suggests that Cl− movement over the plasma membrane of lymphatic myocytes is integral for spontaneous and α-adrenoceptor-evoked contractility in human collecting lymphatic vessels. Consistent detection and localization of TMEM16A to myocytes suggests that this channel could play a major functional role. NEW & NOTEWORTHY In this study, we report the first observations of Cl− being a critical ionic component of spontaneous and agonist-evoked contractility in human lymphatics. The most consistently expressed Ca2+-activated Cl− channel gene in the human thoracic duct and mesenteric lymphatic vessels appears to be transmembrane member 16A, suggesting that this channel plays a major role.

Voltage-gated sodium channels (VGSC) play a key role for initiating action potentials (AP) in excitable cells. VGSC in human lymphatic vessels have not been investigated. In the present study, we report the electrical activity and APs of small human lymphatic collecting vessels, as well as mRNA expression and function of VGSC in small and large human lymphatic vessels. The VGSC blocker TTX inhibited spontaneous contractions in six of 10 spontaneously active vessels, whereas ranolazine, which has a narrower VGSC blocking profile, had no influence on spontaneous activity. TTX did not affect noradrenaline-induced contractions. The VGSC opener veratridine induced contractions in a concentration-dependent manner (0.1-30 μm) eliciting a stable tonic contraction and membrane depolarization to -18 ± 0.6 mV. Veratridine-induced depolarizations and contractions were reversed ∼80% by TTX, and were dependent on Ca(2+) influx via L-type calcium channels and the sodium-calcium exchanger in reverse mode. Molecular analysis determined NaV 1.3 to be the predominantly expressed VGSC isoform. Electrophysiology of mesenteric lymphatics determined the resting membrane potential to be -45 ± 1.7 mV. Spontaneous APs were preceded by a slow depolarization of 5.3 ± 0.6 mV after which a spike was elicited that almost completely repolarized before immediately depolarizing again to plateau. Vessels transiently hyperpolarized prior to returning to the resting membrane potential. TTX application blocked APs. We have shown that VGSC are necessary for initiating and maintaining APs and spontaneous contractions in human lymphatic vessels and our data suggest the main contribution from comes NaV 1.3. We have also shown that activation of these channels augments the contractile activity of the vessels.

BACKGROUND: Mesenteric lymphatic vessels (MLVs) from various animal species have been intensively studied. We aimed to establish the viability and basic contractile characteristics of human MLVs maintained in vitro and to determine the reactivity of MLVs with norepinephrine (NE) and substance P (SP) and to compare with the thoracic duct (TD).METHODS AND RESULTS: Isolated human lymphatic vessels were mounted on a wire myograph under isometric conditions and tension was recorded. The diameter-tension characteristics for MLVs were generated by stretching the vessels and stimulating with a 125 mM K(+) solution containing 10 μM NE. The diameter-tension data generated for MLVs from two separate surgical patient groups were found to be similar: maximum active tension for MLVs occurred when the passive stretch corresponded to a transmural pressure of 22 mmHg. Subsequent experiments on human MLVs were performed by normalization with 22 mmHg as the equivalent target pressure. The majority of MLVs were responders (spontaneous activity and/or reactivity with 10 μM NE or 125 mM K(+) solution). Nonresponders (16% of vessel segments) had significantly smaller inner diameters. MLVs responded consistently to NE (1 nM-10 μM) but the responsiveness of MLVs and TD to SP (0.1 nM-10 μM) was poor: TD reacted only with 10 μM SP, whereas MLVs were sensitive to nanomolar concentrations and the contractile response declined with higher concentrations.CONCLUSIONS: Under in vitro isometric conditions, human MLVs generate maximum tension when stretched to a passive level corresponding to 22 mmHg, and the majority of MLVs are responsive when normalized to this pressure. MLVs respond to NE and SP though NE produces a more consistent response in the concentration range tested.