Neuropeptide modulation of lymphatic smooth muscle tone in the canine forelimb

Neurokinin A and B are putative inflammatory mediators. We assessed their ability to alter prenodal lymphatic resistance. Intralymphatic neurokinin A (3.0 × 10−6, 3.0 × 10−5 and 3.0 × 10−4 mol l−1) significantly constricted lymphatics at the two highest doses. Preliminary experiments suggested that neurokinin B might dilate lymphatics. To test this, lymphatic pressure was increased by norepinephrine (3.1 × 10−6 mol l−1). Neurokinin B (2.7 × 10−4 mol l−1) was then infused intralymphatically during norepinephrine infusion. Norepinephrine increased perfusion pressure from 5.6 ± 0.6 mmHg to 12.1 ± 1.4 mmHg. Subsequent infusion of neurokinin B significantly decreased lymphatic perfusion pressure from 11.9 ± 1.3 mmHg to 9.9 ± 1.1 mmHg. These data indicate that neurokinin A and B can alter lymphatic resistance and are consistent with the hypothesis that lymph vessel function may be subject to modulation by neurokinins.

lymphatics at the two highest doses. Preliminary experiments suggested that neurokinin B might dilate lymphatics. To test this, lymphatic pressure was increased by norepinephrine (3.1 10 -6 mol 1-1). Neurokinin B (2.7 10 -4 mol 1-1) was then infused intralymphatically during norepinephrine infusion. Norepinephrine increased perfusion pressure from 5.6 0.6mmHg to 12.1 _+ 1.4 mmHg. Subsequent infusion of neurokinin B significantly decreased lymphatic perfusion pressure from 11.9 + 1.3 mmHg to 9.9 + 1.1 mmHg. These data indicate that neurokinin A and B can alter lymphatic resistance and are consistent with the hypothesis that lymph vessel function may be subject to modulation by neurokinins.

Introduction
Neurokinin A (also known as substance K) and neurokinin B (also known as neuromedin K) are both ten amino acid peptides of the tachykinin family which were originally isolated from mammalian spinal cord by Kimura et al. in 1983. The distribution of the neurokinins in both the central and peripheral nervous systems has been reported to be similar to that of substance P, the prototype of the tachykinin family. 2 The concentration of these peptides in perivascular nerve terminals impinging upon the resistance vessels of the peripheral circulation has led to the suggestion that they play a role in the modulation of the circulation. 3'4 Additionally, evidence is accumulating which suggests that alterations in tachykinin levels may play a role in a multitude of pathophysiological conditions. 3'sq We have previously shown that the intra-arterial infusion of nanogram quantities of neurokinin A and B results in potent vasodilation in the skin and skeletal muscle circulations of the canine forelimb.
Although the vascular actions of these neuropeptides have begun to be delineated, no work has yet been published concerning the possible impact of these agents on lymphatic vessel function via alterations in lymphatic smooth muscle tone.
Previous reports have revealed that the central lacteal lymphatics of canine ileal and duodenal villi 12'13 and the lymphatic capillaries in rat liver 4 are innervated with nerves which contain tachykinins. These findings indicate that these vessels (C) 1992 Rapid Communications of Oxford Ltd might be subject to control, at least in part, by tachykinins.
A wide array of endogenous vasoactive agents including catecholamines, 5 autacoids, 16 inflammatory mediators, 16 prostanoids 17 and endothelin 8 are capable of increasing lymphatic resistance when administered either intralymphatically or intraarterially. In the current study we assessed the ability of intralymphatically infused neurokinin A and B to alter lymphatic smooth muscle tone in perfused prenodal lymphatic vessels.

Materials and Methods
Adult mongrel dogs of either sex were anaesthetized with sodium pentobarbital (35 mg kg -1 i.v. and supplemented as needed), intubated and ventilated with room air. Small incisions were made in the skin of the right forelimb and the brachial artery. A small side branch of the brachial artery and a skin small vein and artery in the paw were isolated. The forelimb was perfused at constant arterial inflow via the brachial artery with blood obtained from a cannulated femoral artery.
Forelimb perfusion pressure was measured from the side branch of the brachial artery, and skin small vein and artery pressures were measured on the dorsal and ventral surfaces of the paw respectively. Systemic arterial pressure was measured by inserting a catheter into the brachial artery and advancing it into the aorta. A catheter was inserted into the left external jugular vein and advanced to the level of the right atrium for measurement of response to neurokinin B had been obtained, the central venous pressure, lymphatic was again perfused with control perfusate A lymph vessel on the dorsal surface of the paw while the intra-arterial infusion of norepinephrine was cannulated in the direction of normal lymph continued. This manoeuvre was performed to flow. The lymph vessel was perfused at constant ensure that the decrease in lymphatic perfusion flow at a volume flow rate of 0.034 ml min -1 with pressure seen during neurokinin B infusion was a perfusate which was the supernatant of a 1"1 indeed caused by neurokinin B and not merely a mixture of autologous arterial blood and hepardissipation of the effects of norepinephrine. When inized Krebs solution. Lymphatic perfusion preslymphatic perfusion pressure had again reached a sure was measured from the perfusion system at a steady state, the intra-arterial infusion of norpoint upstream to the point of cannulation of the epinephrine was discontinued and lymphatic prenodal lymph vessel. A three-way stopcock, perfusion pressure was allowed to return to control which had been modified such that all three ports values.
were confluent, allowed for measurement of The effects of a single dose of substance P, the lymphatic perfusion pressure and for the lymphatic prototype of the tachykinin peptide family, was also to be perfused with either control perfusate or tested for its ability to alter lymphatic resistance. perfusate containing tachykinins.
Following acquisition of control values, substance The protocol was as follows" in all experiments, P at a concentration of 2.4 10 -4 mol 1-1 was the lymph vessel was perfused with control infused intralymphaticallyforaminimumof15 min perfusate for a minimum of 15 min to ensure that or until the peak response was obtained. Following all measured pressures had reached steady state the conclusion of the substance P infusion, the values. In the neurokinin A experiments, the lymph lymphatic vessel was again perfused with control vessel was then perfused with a solution containing perfusate until all measured pressures returned to neurokinin A at 3.0 10 -6 3.0 x 10 -5 or control values.
3.0 10 -4 mol 1-1 for a minimum of 15 min or Norepinephrine, neurokinin A, neurokinin B and until the peak response in lymphatic perfusion substance P were made up fresh daily in normal pressure had been obtained. The lymphatic was saline in a stock solution of 1.5 mg m1-1. Final then perfused with control perfusate until the dilutions of norepinephrine were made in normal lymphatic perfusion pressure returned to control saline and it was infused into the arterial blood values. Early experiments revealed that neurokinin supply to the forelimb with a needle tipped catheter. A shares, with histamine and angiotensin II, the Final dilutions of the tachykinins were made by characteristic of altering lymphatic perfusion adding the appropriate amount of the lymphatic pressure upon its initial infusion but thereafter perfusate. All data were analysed using Student's t renders the lymph vessel insensitive to infusion of test as modified for paired replicates. Pressures the same agent even at concentrations several orders obtained immediately prior to an experimental of magnitude higher. Therefore, the three point manoeuvre were compared with those achieved dose-response relationship to neurokinin A had to during the peak of the response seen during the be generated in three separate (n 7) series of manoeuvre. animals to ascertain the actual potency of this agent in altering prenodal lymphatic resistance.

Results
Preliminary experiments with the intralymphatic infusion ofneurokinin B suggested that this peptide The infusion of neurokinin A at the lowest would likely dilate lymphatic vessels. Since the infusion rate did not significantly alter lymphatic control pressure in these vessels as perfused in this perfusion pressure ( Figure 1) from its control value study is relatively low (4-5 mmHg on average) it of 5.7 + 0.6 mmHg. However, infusion of the appeared best to first increase the lymphatic middle dose ofneurokinin A significantly increased perfusion pressure with the intra-arterial infusion of lymphatic pressure from a control value of a known lymphatic constrictor agent and then infuse neurokinin B intralymphatically in the face of the steady but elevated lymphatic perfusion pressure. To this end, in the neurokinin B experiments, norepinephrine was infused intraarterially at a concentration of 3.1 x 10 -6 mol 1-1.
When the lymphatic perfusion pressure had reached a steady elevated value, neurokinin B was 6.7 -+-0.8 mmHg to a peak pressure of  (Table 1).
In the neurokinin B experiments, the intraarterial infusion of norepinephrine resulted in a significant increase in lymphatic perfusion pressure from a control value of 5.6 -t-0.6 mmHg to a peak pressure of 12.1 -+-1.4 mmHg (Figure 2). Systemic pressure, forelimb perfusion, skin small artery and vein pressures were all significantly increased ( Table  2). The intralymphatic infusion of neurokinin B during the continued infusion of norepinephrine resulted in a significant decrease in lymphatic perfusion pressure from its pre-neurokinin level of 11.9 q-1.3 mmHg to a nadir of 9.9 _+ 1.1 mmHg ( Figure 2). Intralymphatic infusion of neurokinin B did not aflect significantly any of the measured vascular pressures (Table 2). When the lymphatic was again perfused with control perfusate during the continued infusion of norepinephrine, lympha-tic perfusion pressure returned to its pre-neurokinin levels. This indicated that the decrease observed during infusion of neurokinin B was not simply a waning of the effects of norepinephrine on the lymphatic. When the intra-arterial infusion of norepinephrine was terminated, lymphatic perfusion pressure returned rapidly to its prenorepinephrine level.
The intralymphatic infusion of substance P at 2.4 x 10 -4 mol 1-1 (n 6) did not significantly change lymphatic perfusion pressure from its control value of 4-t-0.4mmHg and did not significantly alter any of the measured vascular pressures.

Discussion
The localization of neurokinins in perivascular nerve terminals innervating the peripheral circulation has led to the suggestion that these endogenous peptides may modulate the peripheral circulation under either normal or pathophysiological conditions. However, a role for these potent vasoactive agents in health or disease has not yet been established. The increase in tachykinin levels in the synovial fluid of patients with osteoarthritis and rheumatoid arthritis, 19 and the known role of substance P, the prototype of the tachykinin family, in neurogenic inflammation 2-22 has led to the suggestion that these agents might alter transvascular fluid and macromolecular flux. Gamse and Saria 7 have reported that neurokinin A and B produce plasma protein extravasation in rat abdominal skin and Fuller et al. 23 noted that the neurokinins produce a weal and flare when injected into human skin. However, preliminary experiments conducted in our laboratory indicated that the neurokinins do not increase skin lymph flow or its protein concentration in the canine forelimb as would be expected with agents which increase microvascular permeability. These data are consistent with previously published experiments 24 which indicated The experiments in this study clearly indicate that both neurokinin A and neurokinin B are capable of altering lymphatic resistance. The intralymphatic infusion of neurokinin A results in significant increases in lymphatic resistance with a threshold dose between 3.0 x 10 -6 and 3.0 x 10 -5 mol 1-1.
At the highest infusion rare utilized, neurokinin infusion results in a doubling of lymphatic resistance. We have previously reported that a wide array of vasoactive agents are capable of constrict- ing prenodal lymphatic vessels in the canine forelimb. [15][16][17][18] The threshold concentrations at which significant lymphatic constriction is manifest varies widely among vasoactive agents, from the extremely potent lymphatic constrictor endothelin (threshold concentration between 10 -l and 10 -9 mol 1-1) to markedly less potent agents such as prostaglandin E1 (threshold concentration between 10 -4 and 10 -3 mol 1-1). The current study indicates that the threshold concentration of neurokinin A required to obtain significant lymphatic constriction (10 -6 to 10 -s mol 1-1) lies in the middle range of tested agents and is comparable to that seen with bradykinin, dopamine and acetylcholine. However, precise conclusions as to the potency of neurokinin A as a lymphatic constrictor are difficult to make. Since neurokinins are located in nerve endings, the lymphatic perfusate concentrations of these compounds which are reported in this study to be effective in altering lymphatic smooth muscle tone may not accurately reflect their true effective concentrations at their effector site on the lymphatic smooth muscle. The effective concentrations at the point of release on the lymphatic smooth muscle cell may well be substantially lower than effective circulating concentrations. The current study also indicates that intralymphatic infusion of neurokinin B is capable of producing a significant decrease in lymphatic resistance. This is best seen, in light of the control lymphatic perfusion pressures involved, in preconstricted lymph vessels. In this respect, neurokinin B is similar in action to the fl2-receptor agonist terbutaline 25 and to adenosine which also significantly relax pre-constricted lymphatics. The concentration of neurokinin B which is needed to produce significant relaxation of pre-constricted lymphatic vessels is similar to that required for both adenosine and terbutaline.
Preliminary studies with substance P in this experimental model had suggested that it did not significantly alter lymphatic perfusion pressure. In this study, to clarify that point, we assessed the actions of a single dose of substance P given intralymphatically. Indeed, as suspected, substance P did not significantly alter lymphatic perfusion pressure. Therefore, it appears that while neurokinin A and neurokinin B administered intralymphatically in adequate doses can alter lymphatic resistance, substance P in comparable doses does not. Since substance P, neurokinin A and neurokinin B are known to be the preferred ligands for the NK-1, NK-2 and NK-3 tachykinin receptors respectively, it could be speculated that the prenodal lymph vessels in the canine forelimb contain NK-2 and NK-3 receptors but not NK-1 receptors. However, this cannot be determined by the results of the current study and will require additional experimentation utilizing specific neurokinin-receptor agonists.
As stated previously, a precise physiological role for the neurokinins has not yet been established firmly but several lines of evidence have implicated these peptides in control of the peripheral circulation. Sann et al. 4 noted that the plantar capillary blood flow in rats was significantly less in a leg whose sciatic nerve had been treated with capsaicin (an agent known to deplete nerves of tachykinins) than in the contralateral untreated leg. They thus concluded that capsaicin sensitive peptidergic afferents may contribute to regional blood flow control in the skin. Neurokinins may also play a role in the nonadrenergic noncholinergic excitatory control of the airways. Thompson et al. 26 reported that tachykinin depletion significantly modifies the nonadrenergic, noncholinergic excitatory responses in guinea-pig trachea. A role for the tachykinins in pathophysiological states has also been proposed. Barnes has suggested that these neuropeptides may be involved in neurogenic inflammatory reactions in asthma.
Uchida et al. 27 noting that neurokinin A is released by chemical irritants such as cigarette smoke suggested that neurokinin A and other tachykinins may mediate the subendothelial oedema caused by cigarette smoke. Lundin et al. 28 reported that the severity of carcinoid heart disease was correlated to the degree to which plasma levels of serotonin and tachykinins were elevated. It has been suggested that tachykinin-mediated stimulation of fibroblasts may increase fibrosis of the valves in carcinoid heart disease. Krause et al. reported that the circulating levels of substance P more than triple in haemorrhagic shock. Since neurokinin A and neurokinin B appear to be co-localized with substance P and are released by common stimuli such as capsaicin, it is likely that neurokinin levels would likewise be elevated in shock.
The results of previously published work clearly indicate that neurokinin A and neurokinin B have potent cardiovascular effects. Their potent vasodilatory actions make them candidate modulators of the peripheral circulation in either health or numerous disease states. Additionally, the current study suggests that they may also play a role in control of lymphatic vessel function via alterations in lymphatic smooth muscle tone. By way of their actions to influence the ability of the lymph vessels to transport fluid, these agents could impact transvascular fluid flux and oedema formation. In addition, their potent vasodilatory effects could act to supplement the increase in transvascular fluid and macromolecular flux produced by inflammatory mediators through an increase in blood flow and perfused capillary surface area. However, additional studies will be required before a firm role for the neurokinins can be established. Measurement of circulating levels of neurokinins under various pathophysiological conditions is needed to determine under what conditions these agents are released and to determine what circulating concentrations are obtained. Also, a continued refinement in the development of specific neurokinin receptor agonists, antagonists and specific releasing agents is required to firmly establish the role of these neuropeptides in modulation of the peripheral circulation under a myriad of conditions.