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Am J Physiol Heart Circ Physiol 281: H60-H66, 2001;
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Vol. 281, Issue 1, H60-H66, July 2001

Parathyroid hormone-related protein-(1-34) inhibits intrinsic pump activity of isolated murine lymph vessels

Risuke Mizuno1, Nobuyuki Ono2, and Toshio Ohhashi1,3

1 First Department of Physiology, Shinshu University School of Medicine, Matsumoto; 2 Department of Electronics and Control Engineering, Nagano National College of Technology, Nagano, 381-8550; and 3 Institute of Organ Transplants, Reconstructive Medicine, and Tissue Engineering, Shinshu University Graduate School of Medicine, Matsumoto 390-8621, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Parathyroid hormone-related protein (PTHrP) was originally found as a tumor-derived vasoactive factor and has also been known to produce significant relaxation of vascular smooth muscles. Thus effects of PTHrP-(1-34), a PTH receptor-binding domain, on spontaneous lymphatic pump activity was investigated in isolated pressurized lymph vessels of mice. Low concentrations (1 × 10-10 and 3 × 10-10 M) of PTHrP-(1-34) dilated lymph vessels and reduced the frequency of pump activity, whereas high concentrations (1 × 10-9 to 1 × 10-8 M) of PTHrP-(1-34) caused dilation with cessation of the lymphatic pump activity. Nomega -nitro-L-arginine methyl ester (L-NAME; 3 × 10-5 M) but not indomethacin (1 × 10-5 M) significantly reduced the PTHrP-(1-34)-induced inhibitory responses of the lymphatic pump activity. In the presence of L-NAME (3 × 10-5 M) and L-arginine (1 × 10-3 M), the L-NAME-induced inhibition in the PTHrP-(1-34)-mediated responses was significantly reduced. Glibenclamide (1 × 10-6 M) significantly suppressed the inhibitory responses of the lymphatic pump activity induced by PTHrP-(1-34) and S-nitroso-N-acetyl-penicillamine. The PTHrP-(1-34)-mediated inhibitory responses were significantly reduced by treatment with PTHrP-(7-34) (1 × 10-7 M). These results suggest that PTHrP-(1-34) inhibits spontaneous pump activity of the isolated lymph vessels via PTH receptors and that production and release of endogenous nitric oxide and activation of ATP-sensitive K+ channels in the lymph vessels contribute to the PTHrP-(1-34)-mediated inhibitory responses of the lymphatic pump activity.

mice; nitric oxide; ATP-sensitive K+ channel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE LYMPHATIC SYSTEM plays an important role in regulating the transport of extracellular fluids and macromolecular substances in tissues and organs in that lymph vessels act to return fluid and protein that escapes from the blood capillaries to the systemic circulation. In the pathophysiological condition, the lymphatic system also works as a route for macrophage traffic in immunological responses and metastasis of malignant carcinoma cells. The transport of escaped fluid, protein, and cells is initiated by a transient pressure gradient through the lymph vessels (2, 33). To produce the pressure gradient, the intrinsic spontaneous activity of the collecting lymph vessels also functions as a series of lymphatic pumps that propel the lymph and cells centripetally (13, 22, 24, 25).

It is well known that humoral and neural factors affect the spontaneous pump activity of lymph vessels (21). Vasoactive peptides such as bradykinin and endothelin play especially pivotal roles in the regulation of intrinsic pump activity of isolated bovine collecting lymph vessels (3, 31). Vasoactive intestinal peptide is also confirmed in lymph vessel walls, the activation of which results in a marked inhibition of lymphatic pump activity (21).

Parathyroid hormone-related protein (PTHrP) was originally found as a tumor-derived humoral factor (26). PTHrP is also known to affect the cardiovascular system as an autocrine and/or paracrine factor (32); thus PTHrP reduces systemic blood pressure in vivo and causes relaxation of vascular smooth muscles (8, 10, 20, 27, 35, 37, 39). It may be reasonable to hypothesize that, as it is a macromolecular substance, the tumor cell-derived PTHrP can easily penetrate initial microlymphatics and then inhibit the intrinsic pump activity of collecting lymph vessels. This may contribute to formation of edema in tumor tissues, increasing of hydrostatic pressure in tissue, and dilution of tumor cell-derived substances including cytokines, growth factors, and lymph vessel active substances such as PTHrP. Dilution of PTHrP and other substances (21) that inhibit vasoactivity may facilitate lymphogenous spread of tumor cells by allowing lymph clearance from tumor sites. Increased lymph clearance may provide a route for tumor cells to leave the primary site and metastasize.

There is, however, no report that evaluates the potential effects of PTHrP on the spontaneous lymphatic pump activity. Therefore, we attempted to study the effects of PTHrP-(1-34), a PTH receptor-binding domain (11), on the spontaneous pump activity of murine isolated iliac lymph vessels and then to investigate the detailed mode of action of PTHrP-(1-34) with special reference to endogenous nitric oxide (NO) and ATP-sensitive K+ channels.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The study subjects were male ddY mice (ages 5-7 wk; n = 44; body weight ~30 g) obtained from Japan SLC. The mice were housed in an environmentally controlled vivarium and fed a standard pellet diet with water ad libitum. All experimental protocols were approved by the Animal Ethics Committee of Shinshu University School of Medicine in accordance with the Japanese Physiological Society's "Guide for the Care and Use of Laboratory Animals."

Isolation and cannulation of lymph vessels. The mice were anesthetized with pentobarbital sodium (50 mg/kg ip) and exsanguinated. After an incision was made in the abdomen, the iliac lymph nodes and the efferent lymph vessels were excised and placed on a petri dish containing cold Krebs bicarbonate solution (~4°C). The Krebs solution contained (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 the use of microsurgical instruments and an operating microscope, 44 lymph vessels (maximum diameter 160.8 ± 3.8 µm; length 3 mm) were isolated and transferred to a 10-ml vessel chamber that contained two glass micropipettes and Krebs bicarbonate solution. The murine lymph vessels have one or two smooth muscle layers, which were confirmed by immunofluorescence staining for anti-smooth muscle alpha -actin antibody (19).

After each lymph vessel was mounted on a pipette (proximal) and secured with a nylon suture (diameter ~ 10 µm), the perfusion pressure was raised to 3 cmH2O to flush and clear the lumen from the vessel. The other end of the vessel was then mounted to the outflow micropipette. The proximal (inflow) micropipette was connected with Tygon tubing and a 50-ml syringe. The distal (outflow) micropipette was connected to Tygon tubing to which a stopcock was attached. The Krebs solution, which was bubbled with 5% CO2-95% N2 gas to yield a pH of 7.40 ± 0.01 and a PO2 of ~50 mmHg, was superfused over the vessel. The flow rate of the solution that was superfused extraluminally over the vessels was 12 ml/min throughout the experiments. The high flow rate of the solution enabled us to keep a constant temperature in the vessel chamber and did not affect spontaneous pump activity of the lymph vessels (16). After the cannulation of the lymph vessel, the chamber was transferred to the stage of an intravital microscope (Nikon Microphoto). The lymph vessels were then slowly warmed to 37°C and allowed to equilibrate for 60 min. The perfusion pressure in the vessels was increased to 3-4 cmH2O by elevating the 50-ml syringe connected to the inflow tubing while the outflow tubing was closed (via the stopcock) throughout the experiment. This perfusion pressure is known to be optimal for producing stable spontaneous pump activity of isolated murine lymph vessels (15).

Measurement of lymph-vessel diameter. Images of lymph vessels were obtained through an objective lens (×4), a photo-eyepiece lens (×5), and a monochrome charge-coupled device camera (KCB-270A; KOCOM) and were displayed on a TV monitor (Hamamatsu Photonics). Changes in the diameters of lymph vessels in response to vasoactive agents were manually and automatically measured with a custom-made diameter-detection device (25), calibrated with a stage micrometer (Nikon), and recorded on a videocassette recorder (HR-S100; Victor) and a direct-writing oscillograph (Recti-Horiz-8K; Sanei-Sokki) (16).

Experimental protocols. A single concentration of PTHrP-(1-34) (either 1 × 10-10, 3 × 10-10, 1 × 10-9, 3 × 10-9, or 1 × 10-8 M) was perfused into the vessel chamber for 3 min to construct a single dose-response curve for the agonist in each lymph vessel. Dose-dependent responses of the lymph vessels for PTHrP-(1-34) were obtained in the absence or presence of 3 × 10-5 M Nomega -nitro-L-arginine methyl ester [L-NAME, an inhibitor of NO synthase (NOS)], 3 × 10-5 M L-NAME + 1 × 10-3 M L-arginine, 1 × 10-6 M glibenclamide (a selective blocker of ATP-sensitive K+ channels), 1 × 10-5 M indomethacin (an inhibitor of cyclooxygenase), or 1 × 10-7 M PTHrP-(7-34) (a PTH-receptor antagonist) (18, 36), respectively. Dose-response curves for S-nitroso-N-acetyl-penicillamine (SNAP; 3 × 10-9 to 1 × 10-7 M) were also obtained in the absence or presence of glibenclamide (1 × 10-6 M). The vessels were incubated with inhibitors for 30 min before responses to the vasoactive substances were evaluated.

Drugs. All salts for the Krebs solution (Wako), PTHrP-(1-34), PTHrP-(7-34) (Peptide Institute), L-NAME hydrochloride, L-arginine hydrochloride, indomethacin (Sigma; St. Louis, MO), SNAP (Dojindo), and glibenclamide (RBI) were used in the present study. PTHrP-(1-34) and PTHrP-(7-34) were dissolved into Na2HPO4/citric acid buffer solution (25 mM) as a stock solution (1 × 10-4 M) and stored at -80°C until used. Glibenclamide and SNAP were dissolved into DSMO. The DMSO in the concentration did not exceed 0.0047% in the vessel chamber. The Na2HPO4/citric acid buffer solution and the DMSO in the concentration used in the present study did not affect the spontaneous pump activity of lymph vessels. Concentrations of drugs were expressed as final concentrations in the vessel chamber. All salts and drugs were prepared on the day of the experiment.

Statistics. The PTHrP-(1-34)-induced responses are expressed as the percentage of inhibition of the spontaneous pump activity of lymph vessels. Thus the averaged frequency (times per minute) of lymphatic pump activity during the PTHrP-(1-34)-induced inhibitory response was normalized by the averaged frequency before application of the agonists. Direct effects of inhibitors on maximum diameter (Dmax), minimum diameter (Dmin), and frequency of spontaneous pump activity of lymph vessels are expressed as %Dmax, %Dmin, and %frequency, respectively, and were normalized by each value before the application of drugs. Ejection fraction, a parameter of spontaneous lymph-vessel contraction, was calculated as follows (25, 34): {pi (Dmax/2)2 - pi (Dmin/2)2}/pi (Dmax/2)2. The data are presented as means ± SE of the mean, and n indicates the number of vessels. Significant differences (P < 0.05) were determined by one-way ANOVA as well as Scheffé's post hoc test and/or paired or unpaired Student's t-test as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PTHrP-(1-34)-induced inhibition of spontaneous pump activity of the lymph vessels. The isolated lymph vessels of mice exhibited spontaneous constriction and dilation at an intraluminal pressure of 3-4 cmH2O. The Dmax, Dmin, frequency, and ejection fraction values for the spontaneous pump activity of the lymph vessels were 160.8 ± 3.8 µm, 142.0 ± 3.6 µm, 13.5 ± 0.2 min-1, and 0.22 ± 0.01, respectively; n = 44 for each measurement.

Low concentrations of PTHrP-(1-34) (1 × 10-10 and 3 × 10-10 M) caused slight dilation of the lymph vessels and slightly decreased the rhythm of the lymphatic pump activity. Thus 1 × 10-10 and 3 × 10-10 M PTHrP-(1-34) reduced the frequency of lymphatic pump activity from 14.3 ± 0.3 to 12.9 ± 0.4 min-1 (n = 16; 90.5 ± 1.9%; P < 0.05 vs. measurements before agonist application) and from 14.0 ± 0.3 to 11.3 ± 0.5 min-1 (n = 16; 80.2 ± 3.1%; P < 0.05 vs. measurements before agonist application), respectively. High concentrations of PTHrP-(1-34) (1 × 10-9 to 1 × 10-8 M) induced a marked dilation of the lymph vessels with cessation of spontaneous pump activity (see Fig. 1A): 3 × 10-9 M PTHrP-(1-34)-induced inhibition of the lymphatic pump activity was 24.5 ± 1.9% (n = 16). The second administration of the same concentration of PTHrP-(1-34) (1 × 10-10 to 1 × 10-8 M) was applied to the same lymph vessel 60 min after the first administration of agonist, and the lymph vessels demonstrated marked tachyphylaxis (see Fig. 1B). Therefore, a lymph vessel was isolated from one mouse and used for each protocol in this study.


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  		Fig. 1.   A: representative tracings of dose-dependent responses of parathyroid hormone-related protein [PTHrP-(1-34); 3 × 10-10 to 3 × 10-9 M] to spontaneous pump activity of isolated lymph vessels in mice. B: tracings show significant tachyphylaxis in response of the same lymph vessel to the second application of the same concentration of PTHrP-(1-34) after 60-min wash. Upward and downward oscillations indicate dilation and constriction of the vessels, respectively. up-arrow  and down-arrow , Starting and finishing points, respectively, for PTHrP-(1-34) superfusion.

Effects of L-NAME, L-NAME + L-arginine, and indomethacin on PTHrP-(1-34)-induced inhibition of lymph-vessel pumping activity. L-NAME (3 × 10-5 M) itself significantly constricted the diameters and increased the frequency of lymphatic pump activity. Thus the L-NAME-induced reductions in the %Dmax and %Dmin values for the lymph vessels were 86.0 ± 2.7% and 85.5 ± 2.9% (n = 4; P < 0.05 vs. measurements before L-NAME for each value), respectively. The %frequency of the lymphatic pump activity increased to 117.7 ± 4.2% (n = 4; P < 0.05 vs. measurements before L-NAME) after treatment with L-NAME. Additional treatment with L-arginine (1 × 10-3 M) significantly reversed both L-NAME-induced reduction of the diameters and increment of the frequency: %Dmax, 91.6 ± 2.5%; %Dmin, 91.1 ± 3.1%; and %frequency, 103.6 ± 3.6% (for each value, n = 4; P < 0.05). Figure 2 shows representative recordings of 3 × 10-9 M PTHrP-(1-34)-induced responses of the lymphatic pump activity (see Fig. 2A), the presence of 3 × 10-5 M L-NAME only (see Fig. 2B) and 3 × 10-5 M L-NAME + 1 × 10-3 M L-arginine (see Fig. 2C). Pretreatment with L-NAME (3 × 10-5 M) significantly reversed the PTHrP-(1-34)-induced inhibitory response of the lymphatic pump activity (see Fig. 2B). Simultaneous treatment with 3 × 10-5 M L-NAME and 1 × 10-3 M L-arginine caused reduction of the L-NAME-induced effects on the 3 × 10-9 M PTHrP-(1-34)-mediated inhibitory response (see Fig. 2C), being quite similar to that produced by 3 × 10-9 M PTHrP-(1-34) alone. These results are summarized in Fig. 3. Thus the values for 3 × 10-9 M PTHrP-(1-34)-induced inhibition of the lymphatic pump activity in both the absence and presence of L-NAME (3 × 10-5 M) alone and L-NAME (3 × 10-5 M) + L-arginine (1 × 10-3 M) were 28.0 ± 5.4% (n = 4), 76.6 ± 6.5% (n = 4; P < 0.05 vs. the absence of L-NAME, and P < 0.05 vs. the presence of L-NAME + L-arginine), and 36.8 ± 5.1% (n = 4), respectively. In the presence of 1 × 10-5 M indomethacin, the PTHrP-(1-34)-induced inhibitory response of the lymphatic pump activity was not affected significantly (data not shown).


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  		Fig. 2.   Representative tracings of 3 × 10-9 M PTHrP-(1-34)-induced inhibitory responses of spontaneous pump activity in isolated murine lymph vessels. A: activity in the presence of only PTHrP-(1-34). B: activity in the presence of PTHrP-(1-34) with 3 × 10-5 M Nomega -nitro-L-arginine methyl ester (L-NAME). C: activity in the presence of PTHrP-(1-34) with 3 × 10-5 M L-NAME + 1 × 10-3 M L-arginine.



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  		Fig. 3.   Effects of 3 × 10-5 M L-NAME (black-triangle; n = 4) and 3 × 10-5 M L-NAME + 1 × 10-3 M L-arginine (; n = 4) on PTHrP-(1-34)-induced inhibition of spontaneous pump activity in isolated murine lymph vessels. * and dagger , Significant differences (P < 0.05) from the PTHrP-(1-34)-induced responses in the absence of L-NAME (open circle ; n = 4) and presence of L-NAME + L-arginine (; n = 4), respectively. [PTHrP-(1-34)], PTHrP-(1-34) concentration. Ordinate shows relative inhibition of lymphatic pump activity normalized by the averaged frequency of pump activity before administration of PTHrP- (1-34).

Effects of glibenclamide on PTHrP-(1-34)-induced inhibition of isolated lymph-vessel pump activity. Glibenclamide (1 × 10-6 M) itself significantly constricted the diameters of the lymph vessels. The glibenclamide-induced reductions of %Dmax and %Dmin for the lymph vessels were 89.4 ± 1.0% and 93.0 ± 1.6%, respectively (n = 4 in each group; P < 0.05 vs. measurements before glibenclamide). There was, however, no significant difference in the %frequency of the lymphatic pump activity before and after the treatment with glibenclamide. Figure 4 demonstrates representative tracings of the effect of glibenclamide (1 × 10-6 M) on the 3 × 10-9 M PTHrP-(1-34)-induced inhibition of the lymphatic pump activity. These results are summarized in Fig. 5. Thus the values for 3 × 10-9 M PTHrP-(1-34)-induced inhibition of the lymphatic pump activity in the absence and presence of glibenclamide (1 × 10-6 M) were 28.3 ± 5.4% and 71.8 ± 11.4% (n = 4 in each group; P < 0.05 vs. measurements in the absence of glibenclamide), respectively.


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  		Fig. 4.   Representative tracings of 3 × 10-9 M PTHrP-(1-34)-induced inhibitory responses of spontaneous pump activity in isolated murine lymph vessels. A: responses to PTHrP-(1-34) alone. B: responses to PTHrP-(1-34) with 1 × 10-6 M glibenclamide.



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  		Fig. 5.   Effect of 1 × 10-6 M glibenclamide (; n = 4) on the PTHrP-(1-34)-induced inhibitory response of spontaneous pump activity in isolated murine lymph vessels. *Significant difference (P < 0.05) from PTHrP-(1-34)-induced responses in the absence of glibenclamide (open circle ; n = 4).

Effects of SNAP on pump activity in the absence or presence of glibenclamide. SNAP (3 × 10-9 to 1 × 10-7 M) caused a dose-dependent inhibition of pump activity of the lymph vessels (data not shown). In the presence of glibenclamide (1 × 10-6 M), the SNAP-induced inhibition was significantly reduced. Thus the values for 1 × 10-7 M SNAP-induced inhibition of the lymphatic pump activity in the absence and presence of glibenclamide (1 × 10-6 M) were 22.5 ± 13.7% and 42.3 ± 16.6% (n = 4 in each group; P < 0.05 vs. measurements in the absence of glibenclamide), respectively.

Effect of PTHrP-(7-34) on PTHrP-(1-34)-induced inhibition of spontaneous pump activity of isolated lymph vessels. Pretreatment with only 1 × 10-7 M PTHrP-(7-34) produced no significant effect on the lymphatic pump activity. Figure 6 shows summarized data of the effects of 1 × 10-7 M PTHrP-(7-34) on the PTHrP-(1-34)-induced inhibition of the lymphatic pump activity. Pretreatment with 1 × 10-7 M PTHrP-(7-34) significantly reversed the PTHrP-(1-34)-induced inhibitory responses. Thus the measurements from 3 × 10-9 M PTHrP-(1-34)-induced inhibition of the lymphatic pump activity in the absence and presence of 1 × 10-7 M PTHrP-(7-34) were 31.6 ± 6.9% (n = 4) and 79.9 ± 9.4% [n = 4; P < 0.05 vs. measurements in the absence of PTHrP-(7-34)], respectively.


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  		Fig. 6.   Effect of 1 × 10-7 M PTHrP-(7-34) (; n = 4) on the PTHrP-(1-34)-induced inhibitory response of spontaneous pump activity in isolated murine lymph vessels. *Significant difference (P < 0.05) from PTHrP(1-34)-induced responses in the absence of PTHrP-(7-34) (open circle ; n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The salient findings of the present study are summarized as: 1) PTHrP-(1-34) inhibits spontaneous pump activity of isolated murine lymph vessels, and 2) the production and release of endogenous NO in the lymph vessels and the activation of ATP-sensitive K+ channels in the lymphatic smooth muscles may contribute to the PTHrP-(1-34)-mediated inhibitory responses of the lymphatic pump activity via PTH receptors.

PTHrP-(1-34)-induced inhibitory responses of spontaneous pump activity of isolated murine lymph vessels. Isolated lymph vessels of mice as well as those obtained from the other animals (13, 16, 22, 28) demonstrated a stable spontaneous pump activity at a controlled pressurized condition. The Dmax, Dmin, frequency, and ejection-fraction values for spontaneous pump activity of isolated murine lymph vessels were 160.8 ± 3.8 µm, 142.0 ± 3.6 µm, 13.5 ± 0.2 min-1, and 0.22 ± 0.01, respectively. In contrast, isolated rat mesenteric and iliac lymph vessels exhibited spontaneous pump activity with a frequency of 20-25 min-1 and an ejection fraction of 0.70-0.80 (13, 16); these measurements are significantly greater than those obtained with murine iliac lymph vessels. Murine mesenteric lymph vessels in vivo also exhibit spontaneous pump activity (25) with frequency (13 min-1) and ejection fraction (0.22) values that are quite similar to those obtained with murine iliac lymph vessels in the present experiments.

In the present study, PTHrP-(1-34) at a lower concentration (~1 × 10-10 M) decreased the frequency of spontaneous pump activity of lymph vessels, whereas a marked dilation and cessation of the spontaneous lymphatic pump activity was observed at a higher concentration (~3 × 10-9 M) of PTHrP-(1-34). There was also a marked tachyphylaxis in response to PTHrP-(1-34) in the lymph vessels as well as in isolated blood vessels (10, 20, 35). The spontaneous contractions of isolated murine portal veins were also known to be inhibited by PTHrP-(1-34) (10, 35). In addition, PTHrP-(1-34) caused marked relaxation of isolated aortic, renal, and coronary arteries. (8, 27, 35, 37, 39). These results suggest that PTHrP-(1-34) has similar pharmacological responses in the lymph vessels as in the blood vessels. It may be reasonable to hypothesize that PTHrP-(1-34) regulates an active lymph-transport mechanism as it appears to cause dilation of lymph vessels and reduction in lymphatic pump activity, which may lead to decreased lymph flow and resulting edema of the regional interstitial space.

Production and release of endogenous NO and activation of ATP-sensitive K+ channels contribute to PTHrP-(1-34)-mediated inhibitory responses of lymphatic pump activity. It is well known that NO is an important vasodilator substance in the cardiovascular system (5, 17). In the lymphatic system, NO has also caused relaxation of the lymphatic smooth muscles and reduction of lymphatic pump activity in in vitro and in vivo studies (1, 6, 14, 23, 31, 34, 38, 40). However, there is no report that demonstrates an NO-mediated response in isolated murine lymph vessels. In the present study, the selective NOS inhibitor L-NAME, used in a sufficient concentration (1, 7), significantly reduced the PTHrP-(1-34)-induced negative chronotropic effects of the lymphatic pump activity in mice. In addition, simultaneous treatment with L-NAME and L-arginine (a substrate for NO) significantly reversed the L-NAME-mediated inhibition of the PTHrP(1-34)-induced responses of the lymphatic pump activity. On the other hand, indomethacin, used in a concentration sufficient to inhibit cyclooxygenase activity (14), did not significantly affect the PTHrP-(1-34)-induced inhibition of the spontaneous pump activity of the lymph vessels. These results strongly suggest that murine lymph vessels can produce and release NO in response to PTHrP-(1-34), whereas vasodilator prostanoids are not involved in the PTHrP-(1-34)-mediated inhibitory response. There is an important controversy regarding the PTHrP-induced NO-mediated vasodilation in blood vessels. In isolated perfused rabbit kidneys, PTHrP-(1-34) produced a NO-mediated renal vasodilation (12). On the other hand, NOS inhibitors did not eliminate the PTHrP-(1-34)-induced relaxation of the isolated murine aorta, although the K+-channel blocker tetrabutylammonium significantly inhibited this relaxation (35). The present study is the first demonstration of PTHrP-(1-34)-induced production and release of NO in the lymph vessels. The conclusion may be strongly supported by our additional finding that SNAP, a NO donor, caused inhibition of spontaneous pump activity of lymph vessels that was similar to the inhibition produced by PTHrP-(1-34).

In the present study, L-NAME alone significantly constricted the lymph vessels and increased the frequency of spontaneous lymphatic pump activity. Additional treatment with L-arginine significantly reduced the L-NAME-induced constriction of the lymph vessels and the positive chronotropic effect. These results suggest that isolated murine lymph vessels can produce and release NO in a pressurized condition, which may be related to the flow-mediated production of NO in the lymph vessels. This finding is compatible with experimental data obtained with isolated rat lymph vessels to demonstrate basal release of NO in the pressurized condition (14).

Another important aspect of the present study is the involvement of ATP-sensitive K+ channels in the PTHrP-(1-34)-mediated inhibitory response, because pretreatment with glibenclamide (a selective inhibitor of ATP-sensitive K+ channels) caused significant reduction in the PTHrP-(1-34)-mediated inhibitory response of the lymphatic pump activity. The conclusion is strongly supported by our recent study (16) that activation of ATP-sensitive K+ channels produced spontaneous pump activity in isolated rat mesenteric lymph vessels.

Activation of PTH receptors is related to the PTHrP-(1-34)-mediated inhibitory response. PTH receptors have been classified into three subtypes: PTH1, PTH2, and PTH3. PTH1 and PTH2 receptors exist in a variety of animal tissues (4, 9, 11, 29), whereas the PTH3 receptor has only been observed in fish (30). PTHrP(1-34), which is a binding domain for the PTH receptors, largely binds to the PTH1 receptor rather than the PTH2 receptor (11). In the present study, the PTHrP-(1-34)-induced inhibition of spontaneous pump activity of murine lymph vessels was significantly antagonized by treatment with PTHrP-(7-34), a PTH-receptor antagonist (18, 36). These results suggest that the PTHrP-(1-34)-induced inhibitory response is mediated by activation of PTH receptors in the lymph vessels.


    ACKNOWLEDGEMENTS

The authors express gratitude to Chugai Yakuhin Pharmaceutical for kind donations of PTHrP-(1-34) and PTHrP-(7-34).


    FOOTNOTES

This study was supported by Japanese Ministry of Education, Science, Sports, and Culture Grants-in-Aid for Scientific Research 09877008 and 11470010.

Address for reprint requests and other correspondence: T. Ohhashi, First 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.

Received 22 September 2000; accepted in final form 19 January 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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