INTRODUCTION

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THE EFFECTS OF ENDOTHELIN (ET) on blood vessels have been widely investigated (27). The ETs are known to be a family of peptides, which include endothelin-1, -2, and -3 (ET-1, ET-2, and ET-3) (34). Two ET receptors have also been cloned, the ET_A receptor, which preferentially binds ET-1 (1), and the ET_B receptor, which has an equal affinity for all isoforms of ETs (28). The ET_A and ET_B receptors are expressed on vascular smooth muscles to mediate contraction, whereas endothelial cells mainly express ET_B receptors (4). An activation of the endothelial ET_B receptors causes a release of prostacyclin (PGI₂) (5) or nitric oxide (NO) from the endothelial cells, which results in a marked vasodilation (33).

On the other hand, little information except for the following papers exists regarding potential effects of ETs on the lymphatic vessels. ET-1 induces a contraction of isolated rat mesenteric (8) and isolated porcine tracheobronchial lymph vessels (25) and also increases intraluminal pressure of prenodal lymph vessels in the canine forelimb (6). Cultured human umbilical vein endothelial cells are also known to release ET-1 predominantly into an abluminal compartment rather than an apical (luminal) compartment (30). The findings may suggest a possibility that ET-1 diffuses mainly in the interstitial space and then moves into lymph vessels.

The lymphatic system plays an important role in the overall homeostasis of body fluids. The mechanisms by which those functions are carried out depend on active and passive driving forces, as well as on the rate of lymph production in organs and tissues. The active driving force is due to the intrinsic contractility of the lymph vessels.

Bovine mesenteric lymphatics show spontaneous intrinsic contractions that produce the active driving force for centripetal propulsion of lymph (13, 19). The frequency and amplitude of the spontaneous contractions are modified by nerves (21), humoral factors (2, 31), and mechanical forces (14). Therefore the present study was designed to investigate the mode of action of ET on the spontaneous contractions in the isolated bovine mesenteric lymphatics with special reference to an endogenous NO and vasodilatory prostanoids.

	MATERIALS AND METHODS

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Tissue preparations. One hundred and thirty lymph vessels (2-4 mm in outer diameter) were dissected quickly from 29 fresh mesenteries, usually 20-30 min after the cattle had been slaughtered. Ring segments, 5-mm long, were prepared from the isolated lymph vessels after removal of surrounding adipose and connective tissues.

Measurement of mechanical activity. Each of the lymphatic ring preparations was suspended in a 10-ml organ bath. The organ bath was perfused at a constant rate of 4 ml/min with Krebs-bicarbonate solution. The solution was kept at 37.0 ± 0.5°C through a heat exchanger and aerated with 95% O₂-5% CO₂ to give a pH of 7.4. The composition of the Krebs solution was as follows (in mM): 120.0 NaCl, 5.9 KCl, 25.0 NaHCO₃, 1.2 NaH₂PO₄, 2.5 CaCl₂, 1.2 MgCl₂, and 5.5 glucose. Two thin silk strings were run through the lumen of a ring preparation and formed into two rings. With the use of a dissecting microscope, we inserted the strings gently through the lumen of the ring preparation to avoid damaging the endothelial cells. The presence of the intact endothelial cells was also confirmed histologically by a silver-staining procedure (24) or pharmacologically by an administration of acetylcholine (36). One string was connected to the lever of a force-displacement transducer (Shinko Tsushin UL-10-120, Tokyo, Japan) and the other to the bottom of the organ bath. The isometric tension detected by the transducer was amplified and recorded on a direct-writing oscillograph (Sanei Sokki, 8 K, Tokyo, Japan). The resting tension of each ring preparation was set at 0.2-0.5 g, being optimal for the appearance of spontaneous contractions in the isolated bovine mesenteric lymph vessels (31, 36).

Experimental protocols. All lymphatic ring preparations were allowed to equilibrate for 60-90 min in the oxygenated bathing medium before we began the experiments. After spontaneous contractions reached a constant rate of 1-3 beats/min, the following experimental protocols were carried out.

The effects of ET-1 on the spontaneous contractions were observed in some ring preparations of the isolated bovine mesenteric lymph vessels. ET-1 was added directly to the organ bath in a single dose with a microsyringe. The doses of the drug were expressed in terms of the base and at the final organ bath concentrations. Thus each concentration of ET-1 was washed out between the doses. The concentration of ET-1 was increased separately from 1.0 × 10¹⁰, 3.0 × 10¹⁰, 5.0 × 10¹⁰, and 7.0 × 10¹⁰ to 1.0 × 10⁹ M. A single concentration of ET-1 was administered at 45-min intervals to prevent tachyphylaxis. A concentration-response curve for ET-1 was obtained in each lymphatic ring preparation. Each experiment was completed in up to 3-4 h. Some lymphatic preparations responded repeatedly by an administration of acetylcholine to evaluate time-dependent changes in the mechanical reactivity of the lymphatic endothelial and smooth muscle cells (time control). The mechanical sensitivity of the lymphatic ring preparations was confirmed to be stable over this time period.

When the effect of a certain antagonist on the ET-1-induced response was examined, the lymphatic ring preparations were pretreated for at least 30 min with the antagonist before the responses to ET-1 were observed. In this case, one or two doses of ET-1 were investigated in one lymphatic preparation. Thus each experiment was completed in up to 3-4 h. No significant change in the responses of the ring preparations to acetylcholine was observed over this time period. The concentration-response curves for ET-1 were compared in the lymphatic ring preparations before and after the treatment with BQ-123 (5 × 10⁷ M), a selective ET_A-receptor antagonist. In some experiments, controls were run with no antagonist to observe time-dependent changes in the sensitivity of the lymphatic ring preparations to ET-1. A single dose-response curve for BQ-3020, a selective ET_B-receptor agonist, was also constructed in some lymphatic ring preparations in the same manner as those obtained with ET-1.

The responses to ET-1 and BQ-3020 were also examined in some lymphatic ring preparations from which endothelial cells had been mechanically removed by rubbing the intimal surface with a Krebs-wetted filter paper. The absence of endothelial cells was confirmed histologically by a silver-staining procedure (24) or pharmacologically by an administration of acetylcholine (36).

To examine roles of endogenous prostanoids and NO in the ET-1-induced responses of the lymphatic ring preparations, effects of acetylsalicylic acid (aspirin, 10⁵ M), a cyclooxygenase inhibitor, N-nitro-L-arginine methyl ester (L-NAME, 10⁵ M), a NO synthesis inhibitor, and L-NAME + L-arginine (10⁴ M) on the ET-1-induced responses were investigated in the other endothelium-intact lymphatic ring preparations.

Drugs. The drugs used were the following: endothelin-1 (human) (Peptide Institute, Osaka, Japan), BQ-123 Na, BQ-3020 (Banyu Pharmaceutical, Tsukuba, Japan), acetylcholine chloride (Daiichi Seiyaku, Tokyo, Japan), aspirin, L-NAME hydrochloride, and L-arginine hydrochloride (Sigma, St. Louis, MO).

Statistics. Effects of the agonists on the spontaneous contractions in the lymphatic ring preparations are evaluated with relative changes in the contraction period between the spontaneous contractions. The relative change in the contraction period is defined as T₂/T₁, where T₁ and T₂ are the average of the five contraction periods between spontaneous contractions before administration of the agonist and during the agonist-induced maximal response, respectively. All results in the text, figures, and tables are expressed as means ± SE. The two-tailed Student's t-test for paired or unpaired data or a one- or two-way ANOVA followed by the Scheffé's test was used to investigate for statistical significance between the groups. Differences between the groups were considered significant at P < 0.05.

	RESULTS

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Effects of ET-1 on spontaneous contractions. Administration of ET-1 (10¹⁰-10⁹ M) caused a dose-related acceleration of the contraction period between spontaneous contractions in bovine isolated mesenteric lymphatics (Fig. 1). Higher concentrations of ET-1 (>7 × 10¹⁰ M) induced a small rise of the basal tone in a dose-dependent manner. Repeated administration (3-4 h) of ET-1 (7 × 10¹⁰ M) at 45-min intervals caused no significant reduction in the ET-1-induced positive chronotropic and inotropic effects on the spontaneous contractions (Fig. 2). Such dose-response curves for ET-1 in the lymphatic ring preparations with intact endothelium are summarized in Fig. 4, A and B.

View larger version (52K): [in this window] [in a new window]   Fig. 1.   Typical recordings of dose-related effects of endothelin (ET)-1 (0.1-1 nM) on spontaneous contractions in a ring preparation of isolated bovine mesenteric lymph vessel. Each dose of ET-1 was administrated directly into the organ bath at intervals of 45 min.

View larger version (55K): [in this window] [in a new window]   Fig. 2.   Typical recordings of effects of repeated administration of ET-1 (0.7 nM) on spontaneous contractions in one ring preparation of bovine mesenteric lymph vessel. Administration of ET-1 was repeated at intervals of 45 min.

Effects of BQ-123 on ET-1-induced positive chronotropic effect. Figure 3 shows a representative recording of effects of 5 × 10⁷ M BQ-123 on the ET-1 (5 × 10¹⁰ M)-induced positive chronotropic and inotropic effects on the spontaneous contractions in the lymphatic ring preparations. The effects of BQ-123 on the ET-1-induced positive chronotropic effect in the ring preparations with intact endothelium are summarized in Fig. 4A (n = 6). Pretreatment with BQ-123 (5 × 10⁷ M) caused a significant reduction of the ET-1-induced positive chronotropic effect on the spontaneous contractions (Fig. 4A).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Representative recordings of effects of 5 × 107 M BQ-123, an ETA-receptor antagonist, on ET-1 (0.5 nM)-induced response on spontaneous contractions in lymphatic ring preparations of isolated bovine mesenteric lymphatics. A: no treatment with BQ-123. B: treatment with 5 × 107 M BQ-123.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   A: effects of ETA-receptor antagonist BQ-123 (5 × 107 M) on dose-response curves for ET-1 in lymphatic ring preparations with intact endothelium [, no treatment with BQ-123 (control), n = 6; , treatment with BQ-123, n = 6]. B: effects of mechanical denudation of lymphatic endothelial cells on ET-1-induced positive chronotropic effect on spontaneous contractions [, endothelium intact, n = 6; , rubbing of endothelium, n = 6]. C: effects of BQ-123 (5 × 107 M) on dose-response curves for ET-1 in lymphatic ring preparations without endothelium [, no treatment with BQ-123 (control), n = 6; , treatment with BQ-123, n = 6]. Ordinate shows a relative change in the contraction period between spontaneous contractions expressed in terms of T2/T1, where T1 and T2 are the average of 5 contraction periods before administration of ET-1 and during ET-1-induced maximal response, respectively. Abscissa is concentration of ET-1 in a logarithmic scale. Each value is presented as mean ± SE (vertical bar); * and ** denote P < 0.05 and P < 0.01 vs. closed circles (A and B) or open squares (C), respectively.

The denudation of the lymphatic endothelial cells caused a significant potentiation of the ET-1-induced positive chronotropic effect on the spontaneous contractions (Fig. 4B). However, the denudation per se produced no significant effect on the basal frequency of spontaneous contractions in the ring preparations (Table 1).

The effects of BQ-123 on the ET-1-induced chronotropic effect were also examined in the endothelium-denuded ring preparations (n = 6) (Fig. 4C). Pretreatment with BQ-123 (5 × 10⁷ M) significantly reduced the ET-1-induced chronotropic effect in the lymphatic ring preparations without endothelium (Fig. 4C). An administration of 5 × 10⁷ M BQ-123, however, caused no significant effect on the basal frequency of spontaneous contractions in the lymphatic ring preparations (Table 1).

Effects of BQ-3020 on spontaneous contractions. Figure 5 demonstrates representative recordings of effects of a specific ET_B agonist (BQ-3020) on the spontaneous contractions in the lymphatic ring preparations with or without intact endothelium. In Fig. 5A, BQ-3020 at a concentration of 5 × 10⁸ M caused a marked transient increase in the contraction periods and decrease in the amplitude of spontaneous contractions in the lymphatic ring preparations with the endothelium. In Fig. 5B, mechanical denudation of the lymphatic endothelial cells caused a significant inhibition of the BQ-3020-induced negative chronotropic and inotropic effects on spontaneous contractions.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Typical recordings of effects of BQ-3020 (50 nM) on spontaneous contractions in lymphatic ring preparations of isolated bovine mesenteric lymphatics with (A) and without (B) endothelium.

Such effect of BQ-3020 on the contraction period between spontaneous concentrations is summarized in Fig. 6. BQ-3020 at concentrations ranging from 10⁸ to 10⁷ M caused a dose-dependent increase in the contraction period between spontaneous contractions in the lymphatic ring preparations with intact endothelium (relative ratio of the contraction periods before to after the administration of BQ-3020: 1.03 ± 0.02 at 10⁸ M BQ-3020 and 1.39 ± 0.07 at 10⁷ M BQ-3020, n = 8; P < 0.05). The endothelial denudation in the lymphatic ring preparations caused a significant reduction of the dose-response curve for BQ-3020 (n = 8) (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Effects of removal of endothelium on BQ-3020-induced increase of contraction period between spontaneous contractions in lymphatic ring preparations of isolated bovine mesenteric lymphatics. Ordinate shows same item as that in Fig. 4. Abscissa is concentration of BQ-3020 in a logarithmic scale. Each value is presented as mean ± SE (vertical bar). * P < 0.05 and ** P < 0.01, endothelium intact (, n = 8) vs. denuded group (, n = 8).

Effect of aspirin, L-NAME, and L-NAME + L-arginine on ET-1-induced positive chronotropic effect. Pretreatment with 10⁵ M aspirin did not alter the ET-1-induced positive chronotropic effect on the spontaneous contractions in the lymphatic ring preparations (n = 6) (Table 2). On the other hand, the ET-1-induced positive chronotropic effect on spontaneous contractions in the lymphatic ring preparations was significantly potentiated by the pretreatment with 10⁵ M L-NAME, and an additional treatment with 10⁴ M L-arginine significantly inhibited the L-NAME-induced potentiation of the ET-1-induced chronotropic effect (n = 6) (Fig. 7).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effects of N-nitro-L-arginine methyl ester (L-NAME, 105 M) () and L-NAME (105 M) + L-arginine (104 M) () on ET-1-induced decrease in contraction period between spontaneous contractions in lymphatic ring preparations of isolated bovine mesenteric lymphatics with intact endothelium (n = 6). Ordinate and abscissa show same items as those in Fig. 4. Each value is presented as mean ± SE (vertical bar). * P < 0.05 and ** P < 0.01, significantly different from control group ();  P < 0.05 and  P < 0.01, significantly different from L-NAME group ().

5

4

	DISCUSSION

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The transport of lymph depends on the passive and active driving forces as well as the rate of lymph production in organs and tissues (19). It is clear that the architecture of lymph vessels with the presence of valves allows lymph transport to occur by passive driving forces such as external compression of the vessels by adjacent tissue (32). The active driving mechanism, which may have a significant role in the centripetal propulsion of lymph, is due to the intrinsic spontaneous contractions of the lymph vessels (11, 19). In bovine mesenteric lymphatics in which smooth muscles are well developed in the wall (20), the frequency and amplitude of the spontaneous contractions significantly affect the active driving forces (18, 19). The frequency and amplitude of the spontaneous contractions in the bovine mesenteric lymph vessels are known to be regulated by several vasoactive substances, such as norepinephrine, 5-hydroxytryptamine, histamine, acetylcholine, prostaglandins (PGs), and bradykinin (13, 18, 36). Especially low concentrations of acetylcholine produce negative chronotropic and inotropic effects on the spontaneous contractions in the bovine mesenteric lymphatics, the responses of which may be mediated by NO released from the endothelial cells through activation of low-affinity muscarinic receptors (36).

ET-1 is a very potent and long-acting vasoconstrictor that is mainly produced by endothelial cells of blood and lymph vessels (25, 34). Reeder and Ferguson (25) reported that ET-1 induced tonic contractions in isolated porcine tracheobronchial lymph vessels that were attenuated by ET_A and/or ET_B blockers. No study, however, had been carried out to investigate the effects of ETs on the spontaneous contractions of isolated lymph vessels in vitro.

In the present experiments, ET-1 produced a dose-dependent decrease of the contraction period between spontaneous contractions in bovine isolated mesenteric lymphatics, which were attenuated by the pretreatment with BQ-123 (Fig. 4A). The same concentration of BQ-123 also significantly reduced the ET-1-induced positive chronotropic effect on the spontaneous contractions in the lymphatic ring preparations without intact endothelium (Fig. 4C). The findings suggest that excitation of ET_A receptors on the lymphatic smooth muscle cells may contribute, in part, to the increase of the frequency of spontaneous contractions in bovine isolated mesenteric lymphatics.

Another important aspect of the present study is that the responses of ET agonists, including ET-1 and BQ-3020, appear to be quite transient and less tachyphylaxic compared with the previous studies of isolated blood and lymph vessels in vitro (8, 25, 34). The difference may be, in part, explained by following the present experimental properties used: 1) we used a specialized organ bath system that was perfused continuously by a Krebs solution at a constant rate of 4 ml/min during the experiments, 2) we directly administered a single dose of ET agonists into the organ bath system to make up dose-response curves for the agonists, and 3) we adopted low concentrations of ET agonists to investigate the effects of the drugs on the spontaneous contractions in the lymphatic ring preparations.

Vascular endothelial cells can release vasodilatory mediators (10), such as NO (23) and/or PGs (3). Stimulation of ET_B receptors on endothelial cells of blood vessels is known to release endogenous NO and/or PGs, which contribute to a relaxation of vascular smooth muscles (5, 33). Acetylcholine-induced endogenous NO-mediated relaxations are shown in the canine isolated thoracic duct (22) and porcine isolated hepatic lymph vessels (12). Histamine and norepinephrine also induce a release of NO from porcine tracheobronchial and mesenteric lymph vessels (26). Recently, rat lymphatic endothelial cells were demonstrated to release both NO and PGs, which regulate the vasomotor activity of the collecting lymph vessels (16).

The present finding that endothelial denudation caused a significant potentiation of the ET-1-induced positive chronotropic effect on the spontaneous contractions (Fig. 4B) may be compatible with a hypothesis that endothelium-derived substances released by ET-1 contribute, in part, to attenuate the chronotropic effect in the lymphatic ring preparations. The hypothesis may be strongly supported by the finding that an ET_B-receptor agonist BQ-3020 (10⁸-10⁷ M) caused a dose-dependent increase of the contraction period between spontaneous contractions in the lymph vessels (Fig. 6). In addition, removal of the lymphatic endothelium caused a significant inhibition of the BQ-3020-induced negative chronotropic effect on spontaneous contractions (Figs. 5 and 6). Pretreatment with 10⁵ M aspirin, the concentration of which is known to inhibit cyclooxygenase significantly in the isolated blood vessels (29), did not affect the ET-1-induced positive chronotropic effect (Table 2), suggesting that prostacyclin and the other prostanoids did not play an important role in the ET-1-induced positive chronotropic effect in the bovine isolated mesenteric lymph vessels.

On the other hand, pretreatment with 10⁵ M L-NAME, the concentration of which was confirmed to inhibit specifically the production of endothelium-derived NO (17), significantly potentiated the ET-1-induced positive chronotropic effect in the lymphatic ring preparations (Fig. 7). Additional treatment with 10⁴ M L-arginine significantly reversed the L-NAME-induced inhibition of the ET-1-induced chronotropic effect. Also, it is well known that endogenous NO produces negative chronotropic and inotropic effects on the spontaneous contractions in the bovine isolated mesenteric lymph vessels (36). These findings and evidence strongly suggest that ET_B receptors may be located on the lymphatic endothelial cells and that the stimulation of the ET_B receptors produces a negative chronotropic effect on spontaneous contractions in the isolated lymph vessels via a release of endogenous NO from the lymphatic endothelial cells.

In conclusion, the present study suggests that there are ET_A receptors on the lymphatic smooth muscle cells and ET_B receptors on the lymphatic endothelial cells in bovine mesenteric lymph vessels. Stimulation of the ET_A receptors elicits the positive chronotropic effect on spontaneous contractions, and stimulation of ET_B receptors induces a release of NO, which results in a decrease of the frequency and amplitude of spontaneous contractions in the lymph vessels.

However, further investigations will be needed in the future to evaluate physiological and pathophysiological roles in low concentrations of ET-induced positive and negative chronotropic effects on the spontaneous contractions in bovine mesenteric lymph vessels. Physiological and pathophysiological roles of endothelins in the blood and the lymphatic system are still unclear. One reason is that circulating endothelin levels in normal and pathological states (1-25 pM) are much lower than the concentrations necessary to elicit contractions of blood vessels in vivo (0.1-50 nM) or produce a positive chronotropic effect on spontaneous contractions in lymph vessels (0.1-1 nM) (9, 15). Because endothelin is preferentially secreted at the abluminal face of the endothelium (30), however, it may accumulate in tissues and lead to local concentrations that are much higher than in the blood circulation. Thus the concentrations of ETs in tissues and lymph in physiological and pathophysiological conditions may be higher than those measured in the plasma. In addition, activated macrophages in inflammation (7) and endothelin-secreting tumors (35) are well known to facilitate expression of mRNA for ET-1 and production of ET-1 protein and then secretion of the ET-1. It may be reasonable to hypothesize that a high concentration of ET in the lymph in the pathophysiological situation such as inflammation or the ET-secreting tumor seems to modulate lymph transport. Low concentrations of ET (0.1-1 nM) in the lymph may lead to increased lymph flow and resulting dehydration of the tissue. On the other hand, high concentrations of ET (>1 nM) in the lymph seem to cause a spasm of lymphatic smooth muscles, which may lead to decreased lymph flow and resulting edema of the tissue.