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Am J Physiol Heart Circ Physiol 287: H2585-H2597, 2004. First published August 12, 2004; doi:10.1152/ajpheart.01080.2003
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ATP inhibits pump activity of lymph vessels via adenosine A1 receptor-mediated involvement of NO- and ATP-sensitive K+ channels

Akira Kousai,1 Risuke Mizuno,1 Fumitaka Ikomi,1 and Toshio Ohhashi1,2

1Department of Physiology, Shinshu University School of Medicine; and 2Institute of Organ Transplants, Reconstructive Medicine, and Tissue Engineering, Shinshu University Graduate School of Medicine, Matsumoto 390-8621, Japan

Submitted 13 November 2003 ; accepted in final form 5 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We examined the effects of ATP on intrinsic pump activity in lymph vessels isolated from the rat. ATP caused significant dilation with a cessation of lymphatic pump activity. Removal of the endothelium or pretreatment with N{omega}-nitro-L-arginine methyl ester (L-NAME) significantly reduced ATP-induced inhibitory responses of lymphatic pump activity, whereas reduction was not suppressed completely by 10–6 M ATP. L-Arginine significantly restored ATP-induced inhibitory responses in the presence of L-NAME. ATP-induced inhibitory responses in lymph vessels with endothelium were also significantly, but not completely, suppressed by pretreatment with glibenclamide. 8-Cyclopentyl-1,3-dipropylxanthine (a selective adenosine A1 receptor antagonist), but not suramine (a P2X and P2Y receptor antagonist) or 3,7-dimethyl-1-proparglyxanthine (a selective adenosine A2 receptor antagonist), significantly decreased ATP-induced inhibitory responses. {alpha},{beta}-Methylene ATP (a selective P2X and P2Y receptor agonist) had no significant effect on lymphatic pump activity. In some lymph vessels with endothelium (24 of 30 preparations), adenosine also caused dose-dependent dilation with a cessation of lymphatic pump activity. L-NAME significantly reduced the inhibitory responses induced by the lower (3 x 10–8–3 x 10–7 M) concentrations of adenosine. Glibenclamide or 8-cyclopentyl-1,3-dipropylxanthine also significantly suppressed adenosine-induced inhibitory responses. These findings suggest that ATP-induced dilation and inhibition of pump activity of isolated rat lymph vessels are endothelium-dependent and -independent responses. ATP-mediated inhibitory responses may be, in part, related to production of endogenous nitric oxide, involvement of ATP-sensitive K+ channels, or activation of adenosine A1 receptors in lymphatic smooth muscle and endothelium.

rat; endothelium; smooth muscle; nitric oxide

THE TRANSPORT OF LYMPH depends on passive and active driving forces as well as the rate of lymph production in organs and tissues. The active driving forces, resulting from intrinsic pump activity of lymph vessels, play a pivotal role in the centripetal propulsion of lymph (1, 31, 39). Neural (21), humoral (30), and mechanical factors (8, 22, 23) modify the rhythm and amplitude of lymphatic intrinsic pump activity. Recently, it has become clear that lymphatic endothelium-derived nitric oxide (NO) strongly contributes to regulation of lymphatic intrinsic pump activity. Thus NO causes relaxation of lymphatic smooth muscles and reduces lymphatic intrinsic pump activity (24, 26, 27, 35, 40, 45, 48).

ATP-sensitive K+ (KATP) channels have been found in the plasma membrane of cells, including vascular and nonvascular smooth muscles, and then became well known to play an important role in regulation of of smooth muscle functions (37). KATP channels in the lymph vessels also play significant roles in control of intrinsic pump activity of the lymphatic smooth muscles (25–27, 44).

Lymphatic smooth muscles in some lymph vessels contain numerous mitochondria and glycogen granules, which may reflect high metabolic activity of lymphatic smooth muscles (32). The existence of blood capillaries within the walls of lymph vessels is related to the high metabolic activity (32). The presence of numerous mitochondria and glycogen granules leads to speculation that a large amount of ATP is produced in the smooth muscles of lymph vessels with intrinsic pump activity. ATP is also an important physiological substance, in that it regulates contraction and relaxation of smooth muscles in the vascular system (3, 38). ATP increased the frequency of spontaneous contractions of lymphatic smooth muscles in sheep (13) and guinea pigs (7, 50). In contrast, isolated and precontracted ring preparations of bovine (33), porcine (10), and canine (41) lymph vessels caused relaxation in response to ATP. Whether ATP can decrease intrinsic pump activity of lymph vessels has not been resolved.

Therefore, in the present study, we have attempted to reexamine the effects of ATP on intrinsic pump activity of isolated rat iliac lymph vessels with special reference to crucial roles of endogenous NO and KATP channels.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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Seven-week-old male Wistar rats (n = 112; Japan SLC) were housed in an environmentally controlled vivarium and fed a standard pellet diet and water ad libitum. All experimental protocols were approved by the Animal Ethics Committee of Shinshu University School of Medicine, in accordance with the principles and guidelines on animal care of the Physiological Society of Japan.

Lymphatic preparations. The rats were anesthetized with pentobarbital sodium (50 mg/kg ip) and exsanguinated. After an incision of the abdomen, the iliac afferent lymph vessels with their lymph nodes were excised and placed in a petri dish containing cold (4°C) Krebs-bicarbonate solution (in mM: 120.0 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 5.5 glucose, and 25.0 NaHCO3). With microsurgical instruments and an operating microscope, the lymph vessels (n = 112, ~250 µm maximum diameter, 3 mm long) were isolated and then transferred to a 10-ml organ chamber with two glass micropipettes containing the Krebs-bicarbonate solution.

After each lymph vessel was mounted on a pipette (proximal) and secured with sutures, the perfusion pressure was raised to 4 cmH2O to flush out and clear the vessel. Then, the distal end of the lymph vessel was mounted to the outflow micropipette (distal). The proximal and distal micropipettes were connected through Tygon tubing with a 50-ml syringe and a stopcock, respectively. The Krebs-bicarbonate solution, in which PO2 was maintained at 56.7 ± 1.6 mmHg (n = 10) and pH at 7.4 ± 0.01 by bubbling with 5% CO2-95% N2 (15, 25–27), was perfused extraluminally over the lymph vessels within the organ chamber. Physiological salt solution with 5% CO2-95% N2 is also useful for studying lymphatic circulation (2, 36, 40, 49), because PO2 in the lymphatic circulation is known to be ~25–40 mmHg, which is lower than that obtained with blood circulation in physiological conditions (9, 16).

The flow rate of the perfused solution was kept at 15 ml/min throughout the experiment. In the present experiments, using the chamber (10 ml) and extraluminal perfusion system (flow rate at 15 ml/min), we needed ≥1 min to obtain maximum concentrations of the drugs in the organ chamber and to wash out the drugs from the chamber (15, 25–27). Thus, to obtain dose-dependent responses of agonists, we circulated extraluminally the Krebs-bicarbonate solution containing each drug into the organ chamber for 3 min. After cannulation of the lymph vessel, the chamber was transferred to the stage of an intravital microscope (Nikon Microphoto). The lymph vessels were then warmed slowly to 37°C and allowed to equilibrate for 60 min.

Measurement of lymph vessel diameter. An objective lens (x4), a photo-eyepiece lens (x3.3), and a monochrome charge-coupled device camera (KOKOM KCB-270A) were used to obtain images of the lymph microvessels, which were displayed on a monochrome television monitor (Hamamatsu Photonics). Changes in the diameter of the lymph vessels were manually and automatically measured with a diameter-detection device with an edge-detection method (36). They were recorded on a videocassette recorder (Toshiba) and a direct-writing oscillograph (Sanei-Sokki, Recti 8K). The intraluminal pressure in the lymph vessels was kept at 5 cmH2O by elevation of a 50-ml syringe connected to the inflow tubing; the outflow tubing was closed with a stopcock throughout the experiments. The pressure was optimal for production of intrinsic pump activity of the isolated rat lymph vessel (15, 23).

Experimental protocols. To evaluate functional viability of the lymphatic endothelial cells, 10–5 M ACh was perfused extraluminally over all the lymph vessels before the experiments were started (15, 23). In the first experimental protocol, to evaluate ATP-induced desensitization of intrinsic pump activity of the lymph vessels, time- and dose-dependent effects of ATP (3 x 10–8–10–6 M, applied 3 times) on lymphatic pump activity were investigated for 30 min. Effects of {alpha},{beta}-methylene ATP (3 x 10–8–10–6 M) on lymphatic pump activity were also investigated. In the second experimental protocol, effects of ATP (3 x 10–8 –10–6 M) on intrinsic pump activity of the lymph vessels with or without intact endothelium were investigated. To remove the lymphatic endothelial cells, air (200 µl) was gently perfused into the lumen of the lymph vessels for 3 min, and then the lumen was flushed with the Krebs-bicarbonate solution for 1 min. The lymph vessels without intact endothelium also exhibited intrinsic pump activity, while the vessels did not show a ACh-induced negative chronotropic effect on lymphatic pump activity (15, 23). To determine histologically the removal of lymphatic endothelium by the air-perfusion method, the lymph vessels were fixed with 10% formalin solution, and the sections (4 µm) were stained with hematoxylin and eosin. In the third protocol, the effects of ATP (3 x 10–8–10–6 M) on lymphatic pump activity were investigated in the absence or presence of 3 x 10–5 M N{omega}-nitro-L-arginine methyl ester (an inhibitor of NO synthase; L-NAME), 3 x 10–5 M L-NAME + 10–3 M L-arginine, 10–5 M indomethacin (a cyclooxygenase inhibitor), or 10–6 M glibenclamide (a selective KATP channel blocker). These concentrations of the inhibitors have been known to produce selectively each specific pharmacological action in the isolated lymph vessels (24–27). In the fourth protocol, the effects of ATP (3 x 10–8–10–6 M) on lymphatic pump activity were investigated in the absence or presence of 10–4 M suramine (an antagonist of P2X and P2Y receptors) (3, 13, 50), 10–7 and 3 x 10–7 M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, a selective adenosine A1 antagonist) (29, 38), or 3 x 10–7 M 3,7-dimethyl-1-proparglyxanthine (DMPX, a selective adenosine A2 antagonist) (29, 38). In the final protocol, the effects of 3 x 10–8–10–6 M adenosine on lymphatic pump activity were investigated in the absence or presence of 3 x 10–5 M L-NAME, 10–6 M glibenclamide, or 3 x 10–7 M DPCPX. The lymph vessels were pretreated with the various blockers and inhibitors for 30 min before extraluminal perfusion of ATP or adenosine through the organ chamber.

Drugs. All salts were obtained from Wako; ACh from Daiichiseiyaku (Tokyo, Japan); glibenclamide, DPCPX, and DMPX from Research Biologicals; and ATP, adenosine, {alpha},{beta}-methylene ATP, L-NAME, L-arginine, indomethacin, and suramine from Sigma. Glibenclamide was diluted with DMSO, and DPCPX, DMPX, and indomethacin were diluted with ethanol. The concentrations of DMSO and ethanol did not affect the myogenic tone and intrinsic pump activity of the isolated lymph vessels. Concentrations of drugs were expressed as the final concentration in the organ chamber. All salts and drugs were prepared on the day of the experiment. ATP, adenosine, and {alpha},{beta}-methylene ATP were kept on ice between applications, which seemed to inhibit degradation of the drugs (34).

Statistical analyses. The frequency (times/min) of intrinsic pump activity and minimum diameter (Dmin, µm) and maximum diameter (Dmax, µm) of the lymph vessels were measured. The drug-induced responses of intrinsic pump activity are expressed as relative changes in the frequency of pump activity and changes in Dmin and Dmax of the lymph vessels. Changes in the parameters during the drug-induced responses were normalized by each control value of the parameters obtained before application of the drugs (15, 27). The drug-induced cessation periods (s) of lymphatic pump activity express the maximum inhibitory time after perfusion of each drug (15). Values are means ± SE, and n indicates the number of vessels. Significant differences (P < 0.05) were determined by one-way ANOVA followed by Student-Newman-Keuls post hoc test and unpaired and paired Student's t-test, as appropriate.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of ATP and {alpha},{beta}-methylene ATP on intrinsic pump activity of lymph vessels. Figure 1A shows representative traces of the effects of 3 x 10–8–10–6 M ATP on intrinsic pump activity of the isolated lymph vessel. ATP caused dose-dependent dilation with a cessation of lymphatic pump activity (Fig. 1, B and C). The dose-dependent responses were obtained with three applications of ATP (3 x 10–8–10–6 M) at 30-min intervals. The percent frequencies of lymphatic pump activity with the first, second, and third applications of 10–6 M ATP were 35.9 ± 6.0, 29.7 ± 5.1, and 23.4 ± 2.3%, respectively (n = 6), and the cessation periods of lymphatic pump activity with the first, second, and third applications of 10–6 M ATP were 173.0 ± 30.8, 164.3 ± 22.6, and 183.8 ± 23.7 s, respectively (n = 6). There was no desensitization with repeated administration of ATP.



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  		Fig. 1. A: representative traces of dose-dependent effects of 3 x 10–8 M-10–6 M ATP on lymphatic intrinsic pump activity in an isolated rat iliac lymph vessel with endothelium. Upward- and downward-pointing arrowheads, beginning and end of drug administration, respectively. B-E: reproducible effects of 3 x 10–8-10–6 M ATP administered 3 times ({circ}, 1st administration; {blacktriangleup}, 2nd administration; {blacksquare}, 3rd administration at 30-min intervals on percent frequency [percentage of frequency (times/min) of lymph pump activity before application of drugs] and cessation period (maximum cessation period of lymph pump activity induced by drugs) of lymphatic pump activity and percent maximum and minimum diameter (%Dmax and %Dmin) of isolated lymph vessels with endothelium (n = 6).

 
ATP significantly increased Dmax and Dmin in a dose-dependent manner, resulting in dilation of the isolated lymph vessels. Thus Dmax and Dmin induced by 10–6 M ATP were 261.5 ± 17.1 µm [P < 0.05 vs. control (241.7 ± 18.3 µm), n = 6] and 261.5 ± 17.1 µm [P < 0.05 vs. control (119.2 ± 15.3 µm), n = 6], respectively (Fig. 1, D and E).

{alpha},{beta}-Methylene ATP (3 x 10–8–10–6 M) caused no significant effect on intrinsic pump activity of the isolated lymph vessels; thus the frequencies of pump activity before and after application of 10–6 M {alpha},{beta}-methylene ATP were 22.7 ± 1.3 and 23.7 ± 1.3 min–1, respectively [104.6 ± 2.5%, n = 6, P = not significant (NS)].

Effects of removal of the endothelium on ATP-induced responses. Figure 2 shows cross-sectional photomicrographs of lymph vessels with and without intact endothelium. The layer of endothelium in the lymph vessel after perfusion with air was completely removed (Fig. 2B).



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  		Fig. 2. Representative photomicrographs of cross sections of hematoxylin-eosin-stained lymph vessels with (A) or without (B) air-rubbing treatment of endothelium. Scale bars, 50 µm. C-F: ATP-induced changes in percent frequency and cessation period of lymphatic pump activity and percent Dmax and Dmin of isolated lymph vessels with ({circ}, n = 7) or without endothelium ({bullet}, n = 7). *Significantly different (P < 0.05) from intact endothelium. Percent frequency and cessation period were determined as described in Fig. 1.

 
The lymph vessels without intact endothelium also exhibited intrinsic pump activity. Average frequencies of pump activity of lymph vessels were 20.0 ± 2.5 and 16.0 ± 1.0 min–1 with and without endothelium, respectively (n = 7, P = NS).

Removal of the endothelium caused a complete reduction of ACh-induced negative ino- and chronotropic effects on lymphatic pump activity (data not shown). In contrast, removal of the endothelium produced a partial reduction of ATP-mediated dilation with a cessation of pump activity of the lymph vessels. Thus the inhibitory response mediated by 3 x 10–7–10–6 M ATP remains in the lymph vessels without endothelium (Fig. 2, C and D). Changes in percent frequency of lymphatic pump activity induced by 3 x 10–7 M ATP with and without endothelium were 43.6 ± 6.0% and 87.9 ± 4.2%, respectively (n = 7, P < 0.05). The cessation periods of lymphatic pump activity induced by 3 x 10–7 M ATP with and without endothelium were 137.9 ± 22.7 and 8.7 ± 2.7 s, respectively (n = 7, P < 0.05).

Changes in percent Dmax mediated by 3 x 10–8–10–7 M ATP were significantly reduced in lymph vessels without intact endothelium (Fig. 2E). Changes in percent Dmin mediated by 10–7–10–6 M ATP were significantly reduced in lymph vessels without intact endothelium (Fig. 2F).

Effects of L-NAME, L-NAME + L-arginine, and indomethacin on ATP-induced responses. Figure 3A shows representative traces of 10–6 M ATP-induced inhibitory responses of lymphatic pump activity without L-NAME, with 3 x 10–5 M L-NAME, or with 3 x 10–5 M L-NAME + 10–3 M L-arginine. The ATP-induced dilation with cessation of lymphatic pump activity was significantly suppressed by pretreatment with 3 x 10–5 M L-NAME. Treatment with L-NAME, however, did not reduce completely the 10–6 M ATP-mediated inhibitory responses. Additional treatment with 10–3 M L-arginine significantly restored the L-NAME-induced reduction of the ATP-mediated inhibitory response (Fig. 3, B and C). Changes in percent frequency of lymphatic pump activity induced by 10–6 M ATP without L-NAME, with L-NAME alone, and with L-NAME + L-arginine were 24.4 ± 3.2% (n = 6), 60.2 ± 7.4% (n = 6, P < 0.05 vs. without L-NAME, P < 0.05 vs. L-NAME + L-arginine), and 20.9 ± 5.2% (n = 6), respectively. The cessation periods of lymphatic pump activity induced by 10–6 M ATP without L-NAME, with L-NAME alone, and with L-NAME + L-arginine were 217.5 ± 25.0 s (n = 6), 45.7 ± 24.4 s (n = 6, P < 0.05 vs. without L-NAME, P < 0.05 vs. L-NAME + L-arginine), and 231.8 ± 49.9 s (n = 6), respectively.



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  		Fig. 3. A: representative traces of effects of 10–6 M ATP on intrinsic lymphatic pump activity with endothelium in the absence of N{omega}-nitro-L-arginine methyl ester (L-NAME, top), presence of 3 x 10–5M L-NAME (middle), or presence of 3 x 10–5 M L-NAME + 10–3 M L-arginine (bottom). See Fig. 1 for an explanation of arrowheads. B-E: effects of 3 x 10–5 M L-NAME ({blacktriangleup}) or 3 x 10–5 M L-NAME + 10–3M L-arginine ({blacksquare}) on ATP-induced changes in percent frequency and cessation period of lymphatic pump activity and percent Dmax and Dmin of isolated lymph vessels with endothelium (n = 6). Percent frequency and cessation period were determined as described in Fig. 1. *Significantly different from absence of L-NAME (P < 0.05). {dagger}Significantly different from L-NAME alone (P < 0.05).

 
There were no significant differences in percent Dmax of the isolated lymph vessels without L-NAME, with L-NAME, or with L-NAME + L-arginine (Fig. 3D). Changes in percent Dmin mediated by 3 x 10–7 M ATP were significantly reduced by L-NAME (Fig. 3E). L-Arginine significantly restored and exacerbated the reduction of percent Dmin mediated by 3 x 10–8 and 10–7 M ATP (Fig. 3E).

There were no significant differences in ATP-induced inhibitory responses of the lymph vessels between the absence and presence of 10–5 M indomethacin. Thus changes in percent frequency and cessation periods of lymphatic pump activity induced by 10–6 M ATP in the presence of 10–5 M indomethacin were 25.0 ± 7.6% [n = 6, P = NS vs. without indomethacin (26.7 ± 10.9%)] and 197.2 ± 50.5 s [n = 6, P = NS vs. without indomethacin (212.7 ± 50.2 s)], respectively. There were no significant differences in percent Dmax and percent Dmin of the isolated lymph vessels between the absence and presence of indomethacin (data not shown).

Effects of glibenclamide on ATP-induced responses. In the presence of 10–6 M glibenclamide, the ATP-induced dilation with a cessation of lymphatic pump activity in the lymph vessels with endothelium was significantly reduced (Fig. 4, A and B). Changes in percent frequency of lymphatic pump activity induced by 10–6 M ATP in the absence and presence of glibenclamide were 28.4 ± 5.3% and 49.4 ± 9.6%, respectively (n = 6, P < 0.05). The cessation periods of pump activity induced by 10–6 M ATP in the absence and presence of glibenclamide were 174.5 ± 30.5 and 103.7 ± 37.4 s, respectively (n = 6, P < 0.05). Thus the 10–6 M ATP-mediated dilation with a cessation of lymphatic pump activity remains in the presence of 10–6 M glibenclamide.



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  		Fig. 4. Effects of 10–6 M glibenclamide ({bullet}) on ATP-induced changes in percent frequency (A) and cessation period (B) of lymphatic pump activity and percent Dmax (C) and Dmin (D) of isolated lymph vessels with endothelium (n = 6). Percent frequency and cessation period were determined as described in Fig. 1. *Significantly different (P < 0.05) from absence of glibenclamide ({circ}).

 
There were no significant differences in percent Dmax (Fig. 4C) and percent Dmin (Fig. 4D) of the lymph vessels between the absence and presence of glibenclamide.

Effects of DPCPX, suramine, or DMPX on ATP-induced responses. In the presence of 10–7 or 3 x 10–7 M DPCPX, the ATP-induced dilation with cessation of lymphatic pump activity in the lymph vessels with endothelium was significantly, but not completely, reduced (Fig. 5, A and B). Changes in percent frequency of lymphatic pump activity induced by 3 x 10–7 M ATP in the absence and presence of 10–7 or 3 x 10–7 M DPCPX were 47.6 ± 9.4% (n = 7) and 73.8 ± 6.0% (n = 7, P < 0.05 vs. without DPCPX) or 89.3 ± 3.6% (n = 7, P < 0.05 vs. without DPCPX), respectively. The cessation periods of lymphatic pump activity induced by 3 x 10–7 M ATP in the absence and presence of 10–7 M or 3 x 10–7 M DPCPX were 148.0 ± 38.3 s (n = 7) and 29.9 ± 17.9 s (n = 7, P < 0.05 vs. without DPCPX) or 13.7 ± 9.3 s (n = 7, P < 0.05 vs. without DPCPX), respectively. There were no significant differences in percent Dmax of the isolated lymph vessels with endothelium between the absence and presence of DPCPX (Fig. 5C). DPCPX (10–7 and 3 x 10–7 M) significantly reduced the 10–7 M ATP-induced increase in percent Dmin (Fig. 5D).



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  		Fig. 5. Effects of 10–7 M ({blacktriangleup}) or 3 x 10–7 M 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, {blacksquare}) on ATP-induced changes in percent frequency (A) and cessation period (B) of lymphatic pump activity and percent Dmax (C) and Dmin (D) of isolated lymph vessels with endothelium (n = 7). Percent frequency and cessation period were determined as described in Fig. 1. *Significantly different (P < 0.05) from absence ({circ}) of DPCPX.

 
Pretreatment with 3 x 10–7 M DPCPX also significantly suppressed 10–6 M ATP-mediated inhibitory responses of lymph vessels without intact endothelium (data not shown).

Suramine (10–4 M) or 3 x 10–7 M DMPX does not suppress the ATP-induced changes in percent frequency and the cessation period of lymphatic pump activity and changes in percent Dmax and percent Dmin of the isolated lymph vessels with endothelium (Figs. 6 and 7). Changes in percent frequency of lymphatic pump activity induced by 10–6 M ATP in the absence and presence of suramine were 25.3 ± 3.6% and 34.4 ± 7.1%, respectively (n = 5, P = NS; Fig. 6A). The cessation periods of pump activity induced by 10–6 M ATP in the absence and presence of suramine were 216.6 ± 17.3 and 213.2 ± 11.8 s, respectively (n = 5, P = NS; Fig. 6B). There were no significant differences in percent Dmax (Fig. 6C) and percent Dmin (Fig. 6D) of the isolated lymph vessels with endothelium between the absence and presence of suramine.



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  		Fig. 6. Effects of 10–4 M suramine ({bullet}) on ATP-induced changes in percent frequency (A) and cessation period (B) of lymphatic pump activity and percent Dmax (C) and Dmin (D) of isolated lymph vessels with endothelium (n = 5). Percent frequency and cessation period were determined as described in Fig. 1. {circ}, Absence of suramine.

 


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  		Fig. 7. Effects of 3 x 10–7 M 3,7-dimethyl-1-proparglyxanthine (DMPX, {bullet}) on ATP-induced changes in percent frequency (A) and cessation period (B) of lymphatic pump activity and percent Dmax (C) and Dmin (D) of isolated lymph vessels with endothelium (n = 6). Percent frequency and cessation period were determined as described in Fig. 1. {circ}, Absence of DMPX.

 
Changes in percent frequency of lymphatic pump activity in the lymph vessels with endothelium induced by 10–6 M ATP in the absence and presence of DMPX were 38.2 ± 7.0% and 32.0 ± 6.3%, respectively (n = 6, P = NS; Fig. 7A). The cessation periods of pump activity induced by 10–6 M ATP in the absence and presence of DMPX were 166.5 ± 46.6 and 154.2 ± 45.5 s, respectively (n = 6, P = NS; Fig. 7B). There were no significant differences in percent Dmax (Fig. 7C) and percent Dmin (Fig. 7D) of the isolated lymph vessels between the absence and presence of DMPX.

Effects of L-LAME, glibenclamide, or DPCPX on adenosine-induced responses. In some lymph vessels (24 of 30 preparations) with endothelium, 3 x 10–8–10–6 M adenosine (Fig. 8) caused dose-dependent dilation with a cessation of lymphatic pump activity; these results were similar to those obtained with ATP (Fig. 1A). In contrast, adenosine produced no significant dilation with a cessation of lymphatic pump activity in some lymph vessels (6 of 30 preparations). With the former lymph vessels, we examined effects of 3 x 10–5 M L-NAME, 10–6 M glibenclamide, or 3 x 10–7 M DPCPX on the adenosine-induced inhibitory responses (Fig. 9).



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  		Fig. 8. Representative traces of dose-dependent effects of 3 x 10–8–10–6 M adenosine on lymphatic intrinsic pump activity with endothelium. See Fig. 1 for an explanation of arrowheads.

 


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  		Fig. 9. Effects of 3 x 10–5 M L-NAME, 10–6 M glibenclamide, and 3 x 10–7 M DPCPX on adenosine (ADO)-induced changes in percent frequency (A) and cessation period (B) of lymphatic pump activity and percent Dmax (C) and Dmin (D) of isolated lymph vessels with endothelium (n = 8). Percent frequency and cessation period were determined as described in Fig. 1. {bullet}, Presence of L-NAME, glibenclamide, or DPCPX; {circ}, absence of L-NAME, glibenclamide, or DPCPX. *Significantly different (P < 0.05) from absence of L-NAME, glibenclamide, or DPCPX.

 
The decreases in percent frequency induced by 3 x 10–7 M adenosine in the absence and presence of 3 x 10–5 M L-NAME were 42.5 ± 6.0% and 75.4 ± 6.8%, respectively (n = 8, P < 0.05; Fig. 9A). The increases in the cessation period induced by 3 x 10–7 M adenosine in the absence and presence of 3 x 10–5 M L-NAME were 179.5 ± 38.9 and 42.0 ± 21.7 s, respectively (n = 8, P < 0.05; Fig. 9B). L-NAME caused no significant effect on the adenosine-induced increase in percent Dmax except at 10–6 M adenosine (Fig. 9C). There were no significant differences in percent Dmin (Fig. 9D) of the lymph vessels between the absence and presence of L-NAME.

The decreases in percent frequency induced by 3 x 10–7 M adenosine in the absence and presence of 10–6 M glibenclamide were 52.7 ± 6.8% and 83.4 ± 3.2%, respectively (n = 8, P < 0.05; Fig. 9A). The increases in cessation period of pump activity induced by 3 x 10–7 M adenosine in the absence and presence of glibenclamide were 146.2 ± 41.3 and 18.0 ± 11.4 s, respectively (n = 8, P < 0.05; Fig. 9B). There were no significant differences in percent Dmax (Fig. 9C) of the lymph vessels between the absence and presence of glibenclamide. Glibenclamide significantly inhibited the adenosine (10–7–10–6 M)-induced increase in percent Dmin (Fig. 9D).

Changes in percent frequency induced by 3 x 10–7 M adenosine in the absence and presence of 3 x 10–7 M DPCPX were 59.4 ± 8.0% and 82.5 ± 5.4%, respectively (n = 8, P < 0.05; Fig. 9A). The increases in the cessation period induced by 3 x 10–7 M adenosine in the absence and presence of 3 x 10–7 M DPCPX were 113.3 ± 39.4 and 27.5 ± 11.6 s, respectively (n = 8, P < 0.05; Fig. 9B). There were no significant differences in percent Dmax of the isolated lymph vessels between the absence and presence of DPCPX (Fig. 9C). DPCPX significantly inhibited the increase in percent Dmin induced by 10–7 M adenosine (Fig. 9D).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The major findings of the present study are summarized as follows. 1) ATP caused dose-dependent dilation with a cessation of intrinsic pump activity in rat isolated lymph vessels. 2) There is no tachyphylaxis. 3) The removal of lymphatic endothelium significantly reduced the ATP-mediated dilation with a cessation of lymphatic pump activity, whereas the reduction was not addressed completely with 10–6 M ATP. Thus the 10–6 M ATP-mediated inhibitory responses remain in lymph vessels without endothelium. 4) L-NAME significantly, but not completely, suppressed the ATP-induced inhibitory responses of lymphatic pump activity in lymph vessels with endothelium. Thus 10–6 M ATP-mediated inhibitory responses remain in the presence of the L-NAME. 5) L-Arginine significantly restored the L-NAME-mediated reduction in the ATP-mediated inhibitory responses. 6) Glibenclamide significantly, but not completely, reduced the ATP-mediated inhibitory responses of lymphatic pump activity in lymph vessels with endothelium. Thus 10–6 M ATP-mediated inhibitory responses remain in the presence of glibenclamide. 7) DPCPX (a selective adenosine A1 antagonist), but not suramine (a P2X and P2Y receptor antagonist) or DMPX (a selective adenosine A2 antagonist), significantly suppressed, but not completely, the ATP-induced inhibitory responses in lymph vessels with or without endothelium. 8) {alpha},{beta}-Methylene ATP had no significant effect on lymphatic pump activity. 9) Adenosine also produced dilation with a cessation of lymphatic pump activity in some lymph vessels with endothelium (24 of 30 preparations). 10) The adenosine-mediated inhibitory responses were significantly, but not completely, reduced by treatment with L-NAME, glibenclamide, or DPCPX. Therefore, we have concluded that ATP-induced dilation and inhibition of intrinsic pump activity of isolated lymph vessels are endothelium-dependent and -independent responses. Production of endogenous NO and activation of KATP channels in lymph vessels contribute, in part, to the ATP-mediated inhibitory responses of lymphatic pump activity in lymph vessels with endothelium.

Involvement of activation of adenosine A1 receptors in the ATP-mediated inhibitory responses. ATP-induced constrictor responses of bovine and guinea pig mesenteric lymph vessels were significantly antagonized by 10–4 M suramine, a blocker of P2X and P2Y receptors (7, 13, 50). The same concentration of suramine used in the present study did not antagonize the ATP-induced inhibitory responses of intrinsic pump activity in these lymph vessels of rats. In contrast, 10–7 and 3 x 10–7 M DPCPX (a selective adenosine A1 receptor antagonist), but not 3 x 10–7 M DMPX (a selective adenosine A2 receptor antagonist), significantly reduced ATP-induced inhibitory responses of lymphatic pump activity in lymph vessels with or without endothelium.

In some lymph vessels with endothelium (24 of 30 preparations), adenosine also caused dose-dependent dilation with a cessation of lymphatic pump activity, which was quite similar to that obtained with ATP in rat iliac lymph vessels with endothelium. Thus the present study also suggests a marked heterogeneity in the adenosine-induced inhibitory responses between the lymph vessels. The adenosine-mediated inhibitory response was also significantly reduced by pretreatment with DPCPX. The concentrations of DPCPX and DMPX used in the present study were known to antagonize selectively the adenosine receptor-mediated responses in in vitro experiments (29).

The ATP-mediated inhibitory responses of the lymph vessels were significantly reduced by removal of the endothelium. In contrast, the ATP-mediated inhibitory responses of lymph vessels were slightly observed after removal of the endothelium (Fig. 2). The 10–6 M ATP-mediated inhibitory responses of lymph vessels without endothelium were significantly reduced by pretreatment with 3 x 10–7 M DPCPX. These findings suggest that ATP may activate, in part, adenosine A1 receptors on the endothelium and smooth muscles of the lymph vessels, resulting in dilation with a cessation of lymphatic pump activity. This conclusion may be compatible with another experimental finding that {alpha},{beta}-methylene ATP had no significant effect on pump activity of the isolated lymph vessels with endothelium.

Adenosine receptors have been classified A1, A2A, A2B, and A3 (38). It is generally accepted that adenosine causes vasodilation via adenosine A2 receptors (38). Adenosine A1 and A2A receptors interact to vasodilate the renal arteriole (28). In addition, there is evidence that differential expression of adenosine receptor subtypes contributes to functional heterogeneity of endothelial cells (6). Further investigation is needed to evaluate the possibility that other adenosine receptors may be involved in ATP-mediated inhibitory responses of lymphatic pump activity. In addition, we should study in the future why adenosine has no significant effect on lymphatic pump activity in some lymph vessels.

Involvement of NO and KATP channels in the ATP-mediated inhibitory responses. NO has an important role in control of physiological functions in the lymph vessels. Thus NO causes relaxation of lymphatic smooth muscles and inhibits intrinsic pump activity of lymph vessels in in vivo and in vitro studies (24, 26, 27, 35, 40, 45, 48). Recently, it has also become clear that NO has a significant role in flow-mediated dilation with a cessation of lymph pump activity (8, 40, 42). KATP channels also significantly contribute to regulation of intrinsic pump activity in lymph vessels (25–27, 44). In the present study, ATP produced dilation with a cessation of lymphatic pump activity in lymph vessels with or without endothelium. The ATP-mediated inhibitory responses were significantly, but not completely, reduced by pretreatment with L-NAME (an NO synthase inhibitor) or glibenclamide (a KATP channel blocker), but not indomethacin (a cyclooxygenase inhibitor). In addition, in some preparations with endothelium, adenosine-mediated inhibitory responses were significantly, but not completely, reduced by pretreatment with L-NAME or glibenclamide. These findings suggest that the ATP- or adenosine-mediated inhibitory responses of lymphatic pump activity are involved, in part, in the production of NO and activation of KATP channels. The findings are quite compatible with the conclusion that adenosine dilated rat diaphragmatic arterioles with involvement of NO production and activation of KATP channels (4).

In the present study, L-arginine significantly restored and also exacerbated the ATP-mediated inhibitory responses in lymph vessels with endothelium (Fig. 3). Extracellular L-arginine is required for optimal NO synthesis by endothelial NO synthase in rat mesenteric artery (20). The concentration of L-arginine may affect the ATP-mediated NO synthase. Thus further investigation is needed to study the effect of L-arginine on the ATP-mediated NO production and/or activity in lymph vessels. However, 10–6 M ATP- or adenosine-mediated inhibitory responses remain in the presence of 3 x 10–5 M L-NAME, 10–6 M glibenclamide, or 3 x 10–7 M DPCPX. Further investigation is needed to evaluate the other mechanisms involved in the inhibitory responses mediated by 10–6 M ATP or adenosine.

ATP-mediated inhibitory responses of lymphatic pump activity. It is well known that myocytes in the heart produce ATP, which is rapidly degraded to ADP, AMP, and adenosine by ectoenzymes. The myocyte-derived adenosine is a key substance in autoregulation of the coronary arterial system (11, 12, 19, 43). Adenosine may play similar crucial roles in the autoregulatory function of the aortic (5), cerebral (14), and renal vasculature (17). Recently, it was reported that cultured pulmonary arterial endothelial cells endogenously produce ATP in response to shear stress and that endogenous release of ATP plays a significant role in the regulation of functions of cultured endothelial cells (47). Immunohistochemical studies indicate the presence of 5'-nucleotidase, an ectoenzyme that converts adenine nucleotides to adenosine, in endothelial cells of lymphatic capillaries and lymph vessels (18, 46, 51). However, a study of metabolism of adenine nucleotides in the lymphatic wall has not been reported.

Lymph vessels exhibit a marked intrinsic contraction and relaxation, which serve as an active pump to transport lymph in vivo and in vitro. ATP increases the frequency of intrinsic pump activity of mesenteric lymph vessels in sheep (13) and guinea pigs (7, 50). The ATP-mediated constrictor response is independent of the function of the endothelium in mesenteric lymph vessels of guinea pigs (50). In the present study, ATP dilated rat iliac lymph vessels and inhibited the frequency of intrinsic pump activity of these vessels. {alpha},{beta}-Methylene ATP, a selective P2X and P2Y receptor agonist for lymph vessels (50), did not affect frequency and amplitude of pump activity of isolated rat afferent lymph vessels. In addition, the ATP-mediated inhibition of rat isolated lymph vessels is an endothelium-dependent and -independent response, in marked contrast to the above-mentioned ATP-mediated excitatory responses of sheep and guinea pig lymph vessels. These findings may indicate a marked heterogeneity, including species and regional differences, receptor subtypes, and intracellular signal transduction mechanisms, in ATP-mediated responses of intrinsic pump activity of lymph vessels. Further investigation is needed to analyze the physiological, pharmacological, and morphological characteristics of the heterogeneity in ATP-mediated responses in the lymph vessels.


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 ABSTRACT
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This study was supported by Japanese Ministries of Education, Science, Sports, and Culture Grants-In-Aid for Scientific Research 13770022, 15700325, 15500315, and 15659310.


   ACKNOWLEDGMENTS
 
The authors are grateful to Dr. Nobuyuki Ono (Nagano National College of Technology) and Yumiko Nakagawa (Shishu University) for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Ohhashi, Dept. of Physiology, Shinshu Univ. School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan (E-mail: ohhashi{at}sch.md.shinshu-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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