Spontaneous and Evoked Contractility of Human Intestinal Lymphatic 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.

Voltage-gated sodium channels contribute to action potentials and spontaneous contractility in isolated human lymphatic vessels.

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.

Human lymphatic vessel contractile activity is inhibited in vitro but not in vivo by the calcium channel blocker nifedipine.

Calcium channel blockers (CCB) are widely prescribed anti-hypertensive agents. The commonest side-effect, peripheral oedema, is attributed to a larger arterial than venous dilatation causing increased fluid filtration. Whether CCB treatment is detrimental to human lymphatic vessel function and thereby exacerbates oedema formation is unknown. We observed that spontaneous lymphatic contractions in isolated human vessels (thoracic duct and mesenteric lymphatics) maintained under isometric conditions were inhibited by therapeutic concentrations (nanomolar) of the CCB nifedipine while higher than therapeutic concentrations of verapamil (micromolar) were necessary to inhibit activity. Nifedipine also inhibited spontaneous action potentials measured by sharp microelectrodes. Furthermore, noradrenaline did not elicit normal increases in lymphatic vessel tone when maximal constriction was reduced to 29.4 ± 4.9% of control in the presence of 20 nmol l(-1) nifedipine. Transcripts for the L-type calcium channel gene CACNA1C were consistently detected from human thoracic duct samples examined and the CaV1.2 protein was localized by immunoreactivity to lymphatic smooth muscle cells. While human lymphatics ex vivo were highly sensitive to nifedipine, this was not apparent in vivo when nifedipine was compared to placebo in a randomized, double-blinded clinical trial: conversely, lymphatic vessel contraction frequency was increased and refill time was faster despite all subjects achieving target nifedipine plasma concentrations. We conclude that human lymphatic vessels are highly sensitive to nifedipine in vitro but that care must be taken when extrapolating in vitro observations of lymphatic vessel function to the clinical situation, as similar changes in lymphatic function were not evident in our clinical trial comparing nifedipine treatment to placebo.

The contribution of K(+) channels to human thoracic duct contractility.

In smooth muscle cells, K(+) permeability is high, and this highly influences the resting membrane potential. Lymph propulsion is dependent on phasic contractions generated by smooth muscle cells of lymphatic vessels, and it is likely that K(+) channels play a critical role in regulating contractility in this tissue. The aim of this study was to investigate the contribution of distinct K(+) channels to human lymphatic vessel contractility. Thoracic ducts were harvested from 43 patients and mounted in a wire myograph for isometric force measurements or membrane potential recordings with an intracellular microelectrode. Using K(+) channel blockers and activators, we demonstrate a functional contribution to human lymphatic vessel contractility from all the major classes of K(+) channels [ATP-sensitive K(+) (KATP), Ca(2+)-activated K(+), inward rectifier K(+), and voltage-dependent K(+) channels], and this was confirmed at the mRNA level. Contraction amplitude, frequency, and baseline tension were altered depending on which channel was blocked or activated. Microelectrode impalements of lymphatic vessels determined an average resting membrane potential of -43.1 ± 3.7 mV. We observed that membrane potential changes of <5 mV could have large functional effects with contraction frequencies increasing threefold. In general, KATP channels appeared to be constitutively open since incubation with glibenclamide increased contraction frequency in spontaneously active vessels and depolarized and initiated contractions in previously quiescent vessels. The largest change in membrane voltage was observed with the KATP opener pinacidil, which caused 24 ± 3 mV hyperpolarization. We conclude that K(+) channels are important modulators of human lymphatic contractility.

The human thoracic duct is functionally innervated by adrenergic nerves.

Lymphatic vessels from animals have been shown to be innervated. While morphological studies have confirmed human lymphatic vessels are innervated, functional studies supporting this are lacking. The present study demonstrates a functional innervation of the human thoracic duct (TD) that is predominantly adrenergic. TDs harvested from 51 patients undergoing esophageal and cardia cancer surgery were either fixed for structural investigations or maintained in vitro for the functional assessment of innervation by isometric force measurements and electrical field stimulation (EFS). Electron microscopy and immunohistochemistry suggested scarce diffuse distribution of nerves in the entire vessel wall, but nerve-mediated contractions could be induced with EFS and were sensitive to the muscarinic receptor blocker atropine and the α-adrenoceptor blocker phentolamine. The combination of phentolamine and atropine resulted in a near-complete abolishment of EFS-induced contractions. The presence of sympathetic nerves was further confirmed by contractions induced by the sympathomimetic and catecholamine-releasing agent tyramine. Reactivity to the neurotransmitters norepinephrine, substance P, neuropeptide Y, acetylcholine, and methacholine was demonstrated by exogenous application to human TD ring segments. Norepinephrine provided the most consistent responses, whereas responses to the other agonists varied. We conclude that the human TD is functionally innervated with both cholinergic and adrenergic components, with the latter of the two dominating.

Human thoracic duct in vitro: diameter-tension properties, spontaneous and evoked contractile activity.

The current study characterizes the mechanical properties of the human thoracic duct and demonstrates a role for adrenoceptors, thromboxane, and endothelin receptors in human lymph vessel function. With ethical permission and informed consent, portions of the thoracic duct (2-5 cm) were resected and retrieved at T(7)-T(9) during esophageal and cardia cancer surgery. Ring segments (2 mm long) were mounted in a myograph for isometric tension (N/m) measurement. The diameter-tension relationship was established using ducts from 10 individuals. Peak active tension of 6.24 +/- 0.75 N/m was observed with a corresponding passive tension of 3.11 +/- 0.67 N/m and average internal diameter of 2.21 mm. The equivalent active and passive transmural pressures by LaPlace's law were 47.3 +/- 4.7 and 20.6 +/- 3.2 mmHg, respectively. Subsequently, pharmacology was performed on rings from 15 ducts that were normalized by stretching them until an equivalent pressure of 21 mmHg was calculable from the wall tension. At low concentrations, norepinephrine, endothelin-1, and the thromboxane-A(2) analog U-46619 evoked phasic contractions (analogous to lymphatic pumping), whereas at higher contractions they induced tonic activity (maximum tension values of 4.46 +/- 0.63, 5.90 +/- 1.4, and 6.78 +/- 1.4 N/m, respectively). Spontaneous activity was observed in 44% of ducts while 51% of all the segments produced phasic contractions after agonist application. Acetylcholine and bradykinin relaxed norepinephrine preconstrictions by approximately 20% and approximately 40%, respectively. These results demonstrate that the human thoracic duct can develop wall tensions that permit contractility to be maintained across a wide range of transmural pressures and that isolated ducts contract in response to important vasoactive agents.