ATP-induced endothelium-independent enhancement of lymphatic vasomotion in guinea-pig mesentery involves P_2X and P_2Y receptors

Abstract

1. The present study has investigated mechanisms underlying ATP-induced endothelium-independent enhancement of vasomotion in guinea-pig mesenteric lymphatic vessels.

2. Lymphatic vasomotion, vessel tone and smooth muscle [Ca²⁺]_i showed similar ATP concentration-response curves.

3. ATP, at 0.1 mM, caused a biphasic increase in tonic [Ca²⁺]_i and superimposed vasomotion-associated Ca²⁺ transients. All ATP-induced [Ca²⁺]_i changes were abolished by incubating the smooth muscle with suramin (0.1 mM).

4. α,β-MeATP (0.1 mM) and UTP (0.1 mM) caused similar changes in [Ca²⁺]_i but the responses to these agonists were smaller than to ATP.

5. The actions of α,β-MeATP (0.1 mM) were inhibited by suramin (0.1 mM) and PPADS (30 μM) but not by reactive blue 2 (30 μM).

6. In the presence of α,β-MeATP (0.1 mM), the increases in tonic [Ca²⁺]_i and vasomotion-associated Ca²⁺ transients induced by ATP (0.1 mM) were inhibited by U73122 (5 μM), CPA (20 μM) and heparin, whereas U73343 (5 μM) and pre-treatment with PTx (100 ng ml⁻¹) had no significant effects.

7. Depletion of the intracellular stores with CPA (20 μM) caused an increase in [Ca²⁺]_i, which was not blocked by desensitization of P_2X receptors with α,β-MeATP.

8. The data indicate that ATP, at relatively high concentrations increases lymphatic smooth muscle [Ca²⁺]_i and vasomotion through activation of P_2X1 and P_2Y2 purinoceptors present on lymphatic smooth muscle. The increase in [Ca²⁺]_i is likely to result from Ca²⁺ release from inositol-1,4,5-trisphosphate-sensitive stores as well as Ca²⁺ influx through store-operated channels and P_2X-gated channels.

Introduction

Adenosine 5′-triphosphate (ATP) has been shown to be a sympathetic co-transmitter in lymphatic vessels where it subserves to modulate rhythmic contractile activity, termed vasomotion (Hollywood & Mchale, 1994). Our previous experiments (Gao et al., 1999) have indicated that extracellular ATP increases vasomotion in guinea-pig mesenteric lymphatic vessels through both endothelium-dependent and endothelium-independent pathways. The former action has been studied in detail and is mainly due to release of the endothelium-derived excitatory factor thromboxane A₂, whereas the mechanisms underlying the endothelium-independent action of ATP on vasomotion remain unknown.

ATP has been shown to cause or modulate vascular constriction through activation of P₂ purinoceptors, receptors that comprise both ligand-gated P_2X ion channel receptors and G protein-coupled P_2Y receptors. These receptors have been further classified at the molecular level (Ralevic & Burnstock, 1998) into various subtypes (P_2X1–7 and P_2Y1,2,4,6). Activation of P_2X receptors leads to an increase in the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) due to a direct Ca²⁺ influx through P_2X receptor-operated channels. α,β-MeATP is a specific agonist of P_2X receptors. P_2X1 receptors have been shown to be the primary P_2X receptor subtypes expressed in vascular smooth muscle (Vulchanova et al., 1996). Binding studies have determined that P_2X1 receptors have a high affinity for ATP and α,β-MeATP (Fredholm et al., 1994). Single channel analysis has shown that these receptors rapidly desensitize in the presence of α,β-MeATP (Surprenant et al., 1995; Werner et al., 1996). In contrast, at least three P_2Y receptor subtypes (P_2Y2, P_2Y4 and P_2Y6) have been reported to be present on vascular smooth muscle where they are involved in modulation of vasoconstriction (Erlinge et al., 1998; Ralevic & Burnstock, 1998). P_2Y1 receptors are also present in blood vessels but are mainly expressed in endothelial cells and contribute to ATP-induced vasodilation (Ralevic & Burnstock, 1998). P_2Y receptors have variable affinities for purine and pyrimidine nucleotides. P_2Y1 receptors are ATP-specific receptors, whereas P_2Y2 receptors have equal affinities for ATP and UTP (Lustig et al., 1993; Nicholas et al., 1996). P_2Y4 receptors are UTP selective, while P_2Y6 are UDP selective and are relatively insensitive to suramin (Boarder & Hourani, 1998; Hartley et al., 1998; Nicholas et al., 1996; Webb et al., 1996).

Lymphatic vasomotion has been indicated to arise through pacemaker Ca²⁺ release within the smooth muscle, causing depolarization and triggering Ca²⁺ action potentials (van Helden, 1993; van Helden, et al., 1996). The present study uses guinea-pig mesenteric lymphatic vessels to investigate the signal pathways underlying the endothelium-independent enhancement of vasomotion induced by ATP. Measurements of smooth muscle [Ca²⁺]_i suggest that extracellular ATP, at higher concentrations than required to activate the endothelium (Gao et al., 1999), enhances vasomotion of lymphatics by direct activation of P_2X1 and P_2Y2 purinoceptors on the smooth muscle. The resultant increases in [Ca²⁺]_i are due to Ca²⁺ influx through P_2X1 ligand-gated channels and P_2Y2-receptor initiated Ca²⁺ release from IP₃-sensitive intracellular stores following activation of the G protein–PLC-IP₃ cascade. In addition, depletion of intracellular IP₃ receptor-operated stores activates a Ca²⁺ influx that is independent of P_2X ligand-gated channels.

Methods

Tissue preparation

Young guinea-pigs (3–10 days) of either sex were killed by an overdose of the inhalation anaesthetic, halothane (5–10% in air) followed by decapitation. Lymphatic vessels (diameter <300 μm) with associated arteries, veins and mesothelium from the ileal region of the intestine were dissected out and pinned flat onto the Sylgard-coated (Dow Corning) base of a small organ bath (volume 1.0 ml). The chamber was continuously superfused at a rate of 6 ml min⁻¹ with a physiological saline solution containing (mM): NaCl 120, KCl 5, CaCl₂ 2.5, MgCl₂ 2, NaHCO₃ 25, NaH₂PO₄ 1 and glucose 10. This solution was maintained at a pH of 7.2 by bubbling with a 95% : 5% O₂ : CO₂ gas mixture and heated to 34–36°C. Perfusion to remove the endothelium and/or to induce vasomotion was effected using a fine glass cannula loosely inserted into the distal end of the lymphatic vessel. The vessel was perfused with a low-calcium physiological saline solution (CaCl₂ 1.2 mM) pumped at a rate of approximately 2.5 μl min⁻¹. Tissues were routinely used within 1–4 h of isolation (except for pertussis toxin experiments) or stored from 2–6 h at 4°C in physiological saline until used.

Ratiometric measurement of [Ca²⁺]_i with Fura 2/AM

Experiments involving measurements of [Ca²⁺]_i changes in the smooth muscle were performed using the calcium sensing dye Fura 2. The smooth muscle of lymphatics was loaded by luminally perfusing endothelium denuded vessels at room temperature with the membrane permeant Fura 2/AM (2 μM) and pluronic acid (0.2%, w v⁻¹) for 30 min. Continued vessel perfusion for a further 10 min washed out the extraneous dye. The tissue was then left for an equilibration time of at least 20 min, allowing the intracellular esterases to cleave Fura 2/AM (Goldman et al., 1990). A metal ring (1×1 cm) bearing the Fura 2 loaded lymphatic vessels and associated arteries, veins and mesentery were placed into an organ bath (volume 0.5 ml) mounted onto an inverted microscope (Zeiss Axiovert 10). The tissue was superfused with physiological saline at a rate of 6 ml min⁻¹ at 34–36°C and viewed through a ×40 oil immersion objective (NA 1.3). Movement artefacts were minimized by performing the experiments without luminal perfusion. The tissue was alternately illuminated with a xenon lamp at wavelengths of 340 and 380 nm for durations of 50 ms for each wavelength with an interval between wavelengths of 50 ms. This cycle was repeated at a frequency of 1–2 Hz. Emission light was passed through a dichroic mirror (490 nm) and a bandpass filter (510 nm) and collected by a photomultiplier with the output response captured by a computer.

Measurement of vasomotion

The constriction of lymphatic vessels was monitored by measurement of either vessel movement or the transient increase in [Ca²⁺]_i in the constricting smooth muscle. Vessel movement was monitored using a video camera attached to an inverted microscope (Nikon Diaphot, ×4 magnification) and analysed visually or videoscopically with vessel edges tracked at 25 Hz by a computer-based algorithm (Diamtrack, (Neild, 1989)). System limitations during Ca²⁺ photometry experiments prevented videoscopic edge-detection. However, measurement made at the isosbestic point of Fura 2 either directly at F₃₆₀ (Furuya et al., 1994) or by calculating F₃₆₀ from F₃₄₀/F₃₈₀ data (Chiavaroli et al., 1994) allowed detection of vessel movement. We found that this movement correlated with the brief Ca²⁺ transients regularly recorded throughout our experiments. These F₃₄₀/F₃₈₀ transients had a rapid rise phase of <0.5 s and duration at half amplitude between 0.5–1.0 s, values commensurate with independent measurements of vessel constriction (van Helden, 1993). Constriction was considered to have occurred when a Ca²⁺ transient of this wave shape presented with amplitudes >1.5 standard deviation of the baseline noise.

Confocal microscopy

Confocal imaging was performed using a BIORAD MRC600 or MRC 1000 laser scanning system attached to an inverted microscope (Zeiss Axiovert 10) with a ×40 oil immersion (NA 1.3) or a ×20 water immersion (NA 0.9) objective. Fluo3 or Oregon loaded tissues were excited at 488 nm using an argon ion laser. Emission fluorescence was collected through a 490 nm dichroic mirror and 515 nm bandpass filter. The AM form of the Ca²⁺ sensitive dye Oregon green (5 μM) was loaded into lymphatic smooth muscle by luminally perfusing endothelium-denuded vessels for 30 min.

Functional removal of endothelium

As described previously (Gao et al., 1999), The endothelium of lymphatics was removed in vitro by repeatedly passing brief streams of air through the lumen of the vessels at a rate of approximately 3 μl min⁻¹. Successful lysis was confirmed by application of acetylcholine (ACh, 100 μM) followed by sodium nitroprusside (SNP, 100 μM). A negative response to ACh and a positive response to SNP were used as confirmation of successful removal of endothelium (von der Weid et al., 1996).

Pertussis toxin incubation

Tissues were superfused for 4 h at 35°C with a physiological saline solution containing pertussis toxin (PTx) (100 ng ml⁻¹) (Burch et al., 1986). Experiments to determine PTx sensitivities were performed subsequent to this incubation but in the absence of PTx in the bathing solution.

Heparin loading

Heparin (8 mg ml⁻¹) was loaded into lymphatic smooth muscle cells using a reversible permeabilization procedure (Kobayashi et al., 1988). Fluo3 (1 μM) salt was simultaneously loaded with heparin to confirm that the tissue had been reversibly permeabilized.

Experimental protocols

Experiments were routinely performed using a 15 min control perfusion period, a 5 min test period with agonists added to the perfusion media, followed by a 30 min washout period. Results were analysed by recording the vasomotion, measured as lymphangion constriction rates, for 4 min at the end of the initial control period, 1 min after application of the agonist and at the end of washout period. When a pharmacological inhibitor was used, the inhibitor was applied for a total of 20 min (15 min before and 5 min during application of agonist). In the case of reversible inhibitors, the antagonist was removed by a 30 min washout period before ATP was again used. Except where noted, analysis has been based on comparisons of the constriction rates of individual lymphangions with respect to the relevant control rates immediately preceding the test condition, both averaged over a 4 min period.

The Ca²⁺ photometry experiments followed the same experimental protocol. Responses were expressed as either a percentage of the control fluorescence ratio immediately preceding the test solution or as the number of Ca²⁺ transients per minute.

Chemicals

Chemicals used were acetylcholine (ACh), adenosine 5′-triphosphate (ATP), heparin, α,β-methylene ATP (α,β-MeATP), sodium nitroprusside (SNP), uridine 5′-triphosphate (UTP) from Sigma-Aldrich; suramin, cyclopiazonic acid (CPA), 1-(6-((17-beta-methoxyestra-1,3,5(10)trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), 1-[6-[[(17beta)-3-Methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2, 5-pyrrolidinedione (U73343) from Biomol Pty Ltd; pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS); reactive blue 2 from Tocris Cookson Ltd; Pertussis toxin and ryanodine from RBI and Fura 2/AM, Fluo 3 and Oregon green/AM from molecular probes. Stock solutions of ACh, ATP, PPADS, reactive blue 2, SNP, suramin, UTP, α,β-MeATP (0.1 M) and Pertussis toxin (100 μg ml⁻¹) were made in distilled water. Stock solutions of CPA, ryanodine (20 mM), U73122, U73343 (5 mM), Fura 2/AM (2 mM) and Oregon green/AM (5 mM) were made in dimethylsulphoxide (DMSO). All stock solutions were stored at −20°C. The final concentration of DMSO used during experiments was always less than 0.1%, a concentration that was found to have no significant effects on lymphatic vasomotion or smooth muscle [Ca²⁺]_i.

Statistical analysis

All constriction frequencies and fluorescence ratios (F₃₄₀/F₃₈₀) were normalized by using the following equation:

where f_N is the normalized value, f is the actual value and f_i is the initial value measured just before application of the test solution. The ATP-induced changes in relative tone (ΔT) were estimated during vasomotion by the following relationship:

Where D is the actual diastolic diameter and D_i is the initial diastolic diameter measured just before application of the test solution.

Dose-response curves were fit by a computer-based program (GraphPad prism 2.01) to the following equation:

where Y is the response at agonist concentration X, Ymin and Ymax are the minimum and maximum responses obtained, EC₅₀ is the concentration giving half-maximal response and H is a Hill coefficient indicating the steepness of the curve.

Data are expressed as the mean±standard error of mean (s.e.mean). Experiments were performed using at least three tissue preparations. Individual n's are stated specifically. Statistical significance was determined using Student's t-test P<0.05 was considered significantly different and P<0.01 highly significantly different for all analyses.

Results

Effect of ATP on lymphatic vasomotion

ATP increased lymphatic vasomotion and tone in perfused lymphatic vessels, as indicated by a respective increase in the frequency of constrictions and decrease in resting diameter (Figure 1). Application of random concentrations of ATP ([ATP]) produced a concentration-dependent increase in both constriction frequency (Figure 2a solid curve) and relative tone, as estimated from vessel diameter ((D_i − D)/D_i; Figure 2b solid curve). Significant increases in both constriction frequency and tone were first observed at [ATP] of 10 nM (110±3.4% of control, P<0.05 and 2.2±1.5% of control, P<0.05; n=12, respectively), with half maximal responses (i.e. EC₅₀) occurring at [ATP] of 17 and 19 μM, respectively (log EC₅₀ −4.8±0.3 and −4.9±0.2). The maximum responses occurred near [ATP] of 3 mM with constriction frequency increasing to 207±12% of control (P<0.01, n=9) and relative tone to 41±3.7% of control (P<0.01, n=9). Removal of the endothelium altered the concentration-response curves (Figure 2a,b, dashed curves), decreasing the maximum responses obtained. The minimum [ATP] required to produce significant increases in lymphatic vasomotion was now significantly higher than for control vessels (i.e. 10 μM vs 10 nM), though the respective EC₅₀ values for vasomotion and tone of 14 and 19 μM (log EC₅₀ −4.9±0.1 and −4.7±0.1) were not significantly different. There was a close link between relative tone and vasomotion in both control vessels (Figure 2c) and endothelium-denuded vessels (Figure 2d).

Figure 1.

The effects of ATP on lymphatic vasomotion in a perfused vessel. Shown are sample traces of ATP-induced enhancement of lymphatic vasomotion and tone at concentrations of 1 μM (a) and 0.1 mM (b).

Figure 2.

The effects of ATP on lymphatic vasomotion and tone in perfused vessels. (a) Concentration–response curves for ATP at concentrations in the range 1 nM–1 mM on lymphangion constriction frequency (i.e. vasomotion) in vessels with or without an endothelium. (b) The corresponding concentration–response plots of the relative change in vessel tone as reflected by the inverse diameter [(D_i−D)/D_i]×100%. Comparison of lymphatic constriction frequency (solid curve) and relative tone [(D_i−D)/D_i] (dashed curve) in vessels with (c) and without (d) endothelium. Data were normalized with respect to the corresponding control lymphangion (chambers formed by two adjacent unidirectional valves (Mislin, 1983)) constriction rates or initial diameters (D_i) with n=9–12 lymphangions. Vertical lines denote s.e.mean.

Effect of ATP on relative [Ca²⁺]_i in lymphatic smooth muscle

ATP was applied to non-perfused, endothelium-denuded vessels loaded with Fura-2 to monitor changes in relative [Ca²⁺]_i. ATP (0.1 mM) caused a transient and significant increase in smooth muscle [Ca²⁺]_i achieving a peak of 154±6.4% of control (P<0.01), which then gradually decreased to a plateau level. The plateau amplitude, measured 1–1.5 min after application of agonist was 141±5.2% of control (P<0.01, n=6). ATP (0.1 mM) also initiated or enhanced oscillatory Ca²⁺ transients (frequency 0.7±0.3 transients/min before and 9±1.9 transients/min after addition of ATP, n=6; Figure 3a). Each transient corresponded to vessel constriction and hence was an indicator of vasomotion (von der Weid et al., 2001).

Figure 3.

The effects of ATP on relative [Ca²⁺]_i in non-perfused lymphatic smooth muscle and the relationship to lymphatic vasomotion in perfused endothelium-denuded vessels. (a) Representative trace showing the effect of 0.1 mM ATP on relative [Ca²⁺]_i. (b) The concentration–response curve for the ATP-induced response on smooth muscle [Ca²⁺]_i. (c,d) Comparisons of relative [Ca²⁺]_i (solid curves) to lymphangion constriction frequency (c, dashed curve) and relative change in vessel tone (d, dashed curve) as reflected by the inverse diameter (D_i−D)/D_i. Data were normalized and expressed as a percentage with respect to the corresponding lymphangion constriction rate, initial diameter or fluorescence ratio of F₃₄₀/F₃₈₀ just before application of ATP with n=6–12 lymphangions for all points. Vertical lines denote s.e.mean.

Applications of random [ATP] (0.1 μM–3 mM) caused concentration-dependent increases in smooth muscle [Ca²⁺]_i with significant responses first detected at an [ATP] of 10 μM (Figure 3b). Comparison of this curve to the corresponding ATP-induced increases in lymphatic constriction frequency and diameter, as obtained in endothelium-denuded vessels (Figure 2), shows close correspondences between both ATP-induced increases in [Ca²⁺]_i and vasomotion (Figure 3c) and relative tone (Figure 3d).

ATP receptor subtypes

Treatment of the tissue with 0.1 mM suramin, a non-specific P₂ purinoceptor antagonist (Dunn & Blakeley, 1988; Hoyle et al., 1990) prevented 0.1 mM ATP-induced increases in both tonic [Ca²⁺]_i (141±8.3% in control and 105±3.3% in suramin, P<0.01, n=6) and the frequency of oscillatory Ca²⁺ transients (8.4±1.4 transients/min in control and 0.8±0.4 transients/min in suramin, P<0.01, n=6). Suramin itself had no significant effects on resting [Ca²⁺]_i or the frequency of the oscillatory Ca²⁺ transients (103±4.8% of control and 1.0±0.4 transients/min, respectively; n=6, P>0.05). Smooth muscle [Ca²⁺]_i responses to ATP appear to involve contributions from both P_2X and P_2Y receptors, as indicated by applications of the P_2X receptor agonist α,β-MeATP (Burnstock & Warland, 1987; Dunn & Blakeley, 1988) and the P_2Y receptor agonist UTP (Nicholas et al., 1996; Webb et al., 1996). α,β-MeATP (0.1 mM) and UTP (0.1 mM) elicited comparable effects, significantly increasing tonic [Ca²⁺]_i to 122±1.0% (n=3, P<0.01, Figure 4a,c) and 127±3.3% of control (n=3, P<0.01, Figure 4b,c) and increasing the frequency of oscillatory Ca²⁺ transients to 4.5±1.0 transients/min (n=3, P<0.01, Figure 4a,d) and 4.1±1.3 transients/min (n=3, P<0.01, Figure 4b,d) respectively. The time course for the [Ca²⁺]_i response to α,β-MeATP (0.1 mM) to return to within 10% of baseline levels was 3.5±0.8 min (n=3). In addition, α,β-MeATP increased vasomotion-associated Ca²⁺ transients as measured using a confocal laser scanning system (Methods). An EC₅₀ of 1.9 μM (n=3–5) was obtained from concentration-response measurements (data not shown). The responses in smooth muscle [Ca²⁺]_i induced by 0.1 mM α,β-MeATP were abolished by 30 μM PPADS (n=3) but not by 30 μM reactive blue 2 (n=3). In the maintained presence of α,β-MeATP (0.1 mM), ATP (0.1 mM) still caused an increase in smooth muscle [Ca²⁺]_i and increased the frequency of oscillatory Ca²⁺ transients (125±2.1% of control and 4±1.0 transients/min respectively; n=4, P<0.01, Figure 5a,c,d). Similar results were obtained when 0.1 mM UTP was applied in the maintained presence of 0.1 mM α,β-MeATP (122±1.5% of control and 4.8±0.9 transients/min respectively; n=4). These data indicate that ATP-induced increases in smooth muscle [Ca²⁺]_i and associated vasomotion are mediated by both P_2X and P_2Y purinoceptors.

Figure 4.

The effects of P_2X and P_2Y agonists on relative [Ca²⁺]_i in lymphatic smooth muscle. Sample traces representing the effects of α,β-MeATP (a) and UTP (b) on the F₃₄₀/F₃₈₀ fluorescence ratio in lymphatic smooth muscle and summary data for these actions compared to the effects of ATP (c,d). Data were normalized with respect to the corresponding fluorescence ratios just before application of either agent. Each measurement is based on data from 3–6 vessels. Vertical lines denote s.e.mean. ^**P<0.01.

Figure 5.

Effects of PTx agonists on relative [Ca²⁺]_i in lymphatic smooth muscle. Sample traces presenting the effects of ATP on smooth muscle fluorescence ratio (F₃₄₀/F₃₈₀) in the presence of α,β-MeATP (a) and α,β-MeATP with PTx (100 ng ml⁻¹, b) and summary data compared to the effects of ATP alone (c,d). Data were normalized with respect to the corresponding fluorescence ratio just before application of ATP or ATP plus other agents. Each measurement represents data from n=3–6 vessels. Vertical lines denote s.e.mean. ^**P<0.01.

Effects of pertussis toxin

Measurements were made to determine whether PTx-sensitive G proteins were involved in the ATP-induced responses, as P_2Y receptors are coupled to G-proteins. Examination of P_2Y receptor-mediated responses, studied in the presence of 0.1 mM α,β-MeATP, demonstrated that the ATP-induced increase of smooth muscle [Ca²⁺]_i and the increase in the frequency of oscillatory Ca²⁺ transients were not abolished by pre-treatment with PTx (amplitude in tonic increase of [Ca²⁺]_i was 117±6.5% of control and the frequency of Ca²⁺ oscillations was 5.0±1.5 transients/min, n=3, Figure 5). These increases were not significantly different to the increases of 125±2% of control and 4.7±1.2 transients/min measured in matched non-PTx treated tissues (n=3). The ability of ATP to cause a response in the presence of α,β-MeATP and PTx indicates that PTx-sensitive G proteins were not involved in the P_2Y receptor-mediated increases in smooth muscle [Ca²⁺]_i and vasomotion.

P_2Y receptor-mediated signalling

The signalling pathways underlying the P_2Y receptor mediated responses were first investigated by testing the effects of inhibiting the PLC-IP₃ pathway. Treatment of lymphatic vessels with U73312 (5 μM, n=3), a blocker of PLC (Thompson et al., 1991), only partially inhibited the ATP-induced increases in smooth muscle [Ca²⁺]_i and frequency of oscillatory Ca²⁺ transients (Figure 6a,b). However, in the presence of a desensitizing concentration of α,β-MeATP (0.1 mM), 5 μM U73122 completely prevented the ATP-induced responses (Figure 6a,b). This was a PLC specific effect, as U73343 (5 μM), an inactive analogue of U73122 (Bleasdale et al., 1990; Thompson et al., 1991) had no significant effects on ATP-induced responses in the presence or absence of 0.1 mM α,β-MeATP (Figure 6a,b).

Figure 6.

The effects of the PLC antagonist U73122 and its inactive analogue U73343 on ATP-induced responses of smooth muscle [Ca²⁺]_i. Shown are the effects of U73122 (5 μM) and U73343 (5 μM) on the 0.1 mM ATP-induced changes in fluorescence ratio (F₃₄₀/F₃₈₀) and oscillatory Ca²⁺ transients (a,b) measured in the absence or presence of α,β-MeATP. All data have been normalized with respect to the control fluorescence ratio just before application of ATP or ATP plus antagonists. n=3–6 for all groups. Vertical lines denote s.e.mean. ^**P<0.01.

A role for IP₃-sensitive Ca²⁺ stores was further investigated using heparin, a specific antagonist of IP₃ receptors (Kobayashi et al., 1988). Heparin (8 mg ml⁻¹) was introduced into smooth muscle cells by a reversible permeabilization procedure (Kobayashi et al., 1988). In the presence of heparin, application of ATP (0.1 mM) caused only a sustained increase in [Ca²⁺]_i without inducing any oscillatory Ca²⁺ transients (n=6; Figure 7). In contrast, the biphasic increase in [Ca²⁺]_i and the frequency of Ca²⁺ transients induced by ATP (n=6, Figure 7a) were still evident in control tissues subjected to the same permeabilization treatment but without heparin. In addition, no evidence for an involvement of ryanodine receptors was found, as application of ryanodine (20 μM) had no significant effects on ATP-induced responses in [Ca²⁺]_i (n=3).

Figure 7.

The effects of heparin on ATP-induced increases in relative [Ca²⁺]_i in lymphatic smooth muscle. Shown are representative traces of changes in the fluorescence ratio (F₃₄₀/F₃₈₀) in response to 0.1 mM ATP in lymphatic vessels subjected to permeabilization treatment of tissues without (a) or with (b) heparin in the external solution. (c) Group data for these responses. Data were normalized with respect to the corresponding control fluorescence ratio just before applications of ATP with n=3–6 for control and treatment groups. Vertical lines denote s.e.mean. ^**P<0.01.

Basis for P₂ purinoceptor mediated mobilization of [Ca²⁺]_i

Extracellular Ca²⁺ entry was found to be a primary contributor to the ATP-mediated increases in [Ca²⁺]_i. The effects of omission of Ca²⁺ from the superfusate (Ca²⁺-free solution with 1 mM EGTA) are shown in Figure 8. The absence of extracellular Ca²⁺ did not block the initial transient increases in [Ca²⁺]_i induced by application of 0.1 mM ATP, but markedly inhibited the maintained increases in [Ca²⁺]_i to 101±3% of control (n=6), a mean value that was not significantly different from baseline levels before addition of ATP (P>0.05). These results suggest that the initial transient increase in [Ca²⁺]_i is predominantly due to release of Ca²⁺ from intracellular Ca²⁺ stores and the sustained component is due to influx of Ca²⁺ from the extracellular space.

Figure 8.

The effect of removal of extracellular Ca²⁺ on the ATP-induced increases in relative [Ca²⁺]_i in lymphatic smooth muscle. Pre-treatment of the tissue with Ca²⁺-free solution containing 1 mM EGTA reduced the normal biphasic increase in relative [Ca²⁺]_i in response to 0.1 mM ATP (a) to a brief transient increase (b).

The sources of Ca²⁺ underlying the ATP-induced [Ca²⁺]_i increases were also investigated by depleting intracellular Ca²⁺ stores using CPA, a blocker of SR Ca²⁺ ATPase (Darby et al., 1993). Incubation of the tissue in CPA (20 μM for 15 min) abolished both the initial transient phase of the ATP-induced increase in [Ca²⁺]_i and the oscillatory Ca²⁺ transients, consistent with the former response arising from Ca²⁺ release from intracellular stores (Figure 9a). In contrast, CPA significantly reduced, but did not abolish the sustained increase in [Ca²⁺]_i observed under control conditions (Figure 9a,c). However, in the presence of 0.1 mM α,β-MeATP (Figure 9b,c), CPA blocked the ATP-induced Ca²⁺ increase, indicating that when store function was interrupted, the residual sustained response was due to extracellular Ca²⁺ entry through P_2X receptor-gated channels. CPA itself caused a small but significant increase in resting [Ca²⁺]_i (121±3.5% of control, n=3, P<0.01, Figure 9d), possibly indicating store depletion-induced Ca²⁺ entry through SOCs. This action was not related to Ca²⁺ entry through P_2X receptor-gated channels since maintained application of α,β-MeATP (0.1 mM) had no significant effect on the CPA-induced increase in resting [Ca²⁺]_i (Figure 9d, n=3).

Figure 9.

The effects of CPA on ATP-induced increases in relative [Ca²⁺]_i in lymphatic smooth muscle. Shown are representative traces of changes in the fluorescence ratio (F₃₄₀/F₃₈₀) induced by 0.1 mM ATP in the maintained presence of 20 μM CPA in the absence (a) and presence (b) of 0.1 mM α,β-MeATP. Summary data for these responses and for the effects of CPA on resting relative [Ca²⁺]_i with and without α,β-MeATP present are shown in (c) and (d) respectively. Data were normalized with respect to the corresponding control fluorescence ratio just before application of the various agents with n=3–6 for all groups. Vertical lines denote s.e.mean. ^**P<0.01.

These data, together with the findings using U73122 and heparin (Figures 6 and 7), provide evidence that the initial increase in [Ca²⁺]_i is due to Ca²⁺ release from IP₃-sensitive Ca²⁺ stores and suggest that the maintained increase in [Ca²⁺]_i is the consequence of Ca²⁺ influx through both P_2X-gated channels and through an internal Ca²⁺ store associated pathway, possibly SOCs.

Discussion

This study investigated pathways by which ATP acts on lymphatic smooth muscle to increase [Ca²⁺]_i and enhance lymphatic vasomotion and tone. Unlike the previously reported endothelium-dependent effects of ATP (Gao et al., 1999), endothelium independent effects occur in response to higher concentrations of ATP (i.e. >1 μM). At these concentrations ATP binds to smooth muscle P_2X and P_2Y purinoceptors, leading to increases in smooth muscle [Ca²⁺]_i by Ca²⁺ influx through P_2X-gated channels and Ca²⁺ release from IP₃-sensitive stores.

Comparison of the ATP concentration–response curves for vasomotion, vessel tone and smooth muscle [Ca²⁺]_i in endothelium-denuded lymphatic vessels showed similar dependencies of these three parameters on [ATP]. This finding indicates that [Ca²⁺]_i is not only a key determinant of vessel tone but also of vasomotion, even though the curves do not perfectly overlie each other. This difference may in part relate to the vasomotion curve here being obtained in perfused vessels and the [Ca²⁺]_i curve from non-perfused vessels as the behaviour of smooth muscle in perfused vessels exhibits some differences to that observed in none perfused vessels. For example vasomotion in perfused vessels does not desensitise as rapidly as in non-perfused vessels (e.g. compare Figure 1 to Figure 3a). We have presented data on both perfused and non-perfused vessels, the former as this represents one extreme of in vivo conditions, when there is massive forward movement of lymph and the latter as it represents the other extreme when there is little movement of lymph. The differences in behaviour between perfused and non-perfused vessels are no doubt complex as the sensitivity of vasomotion to other key agents not dealt with in this paper (e.g. cyclopiazonic acid; Zhao & van Helden unpublished observations) is also different in perfused vessels. This alteration arises in part through perfusion-associated endothelial derived factors, but there seem to be perfusion-associated differences even in endothelium-denuded vessels.

P_2X receptors were found to be involved in the ATP action as the P_2X purinoceptor agonist α,β-MeATP (Fredholm et al., 1994) increased smooth muscle [Ca²⁺]_i as did ATP, the latter applied with the PLC/IP₃ cascade blocked. Lewis & Evans have found that in rat mesenteric artery smooth muscle the P_2X1 receptor-activated current is very rapidly desensitized and has an EC₅₀ of approximately 1–5 μM (Lewis & Evans, 2000b) (also for review see North & Surprenant, 2000). However P_2X1 receptor activated constriction of arteries was demonstrated to follow a time course of about 2–5 min (Lewis et al., 1998; Gitterman & Evans, 2000; Lewis & Evans, 2000a), similar to the results recorded from our present experiments in mesenteric lymphatics and arteries (3.5±0.5 min, n=3, unpublished observations). Further evidence indicating activation of P_2X1 receptors by ATP in mesenteric lymphatics is provided by the finding that α,β-MeATP increased lymphatic vasomotion with a EC₅₀ of 1.9 μM. This is similar to findings in vascular smooth muscle, where the responses have been shown to be P_2X1-mediated responses (Gitterman & Evans, 2000).

The sensitivity to α,β-MeATP and the effectiveness of PPADS and suramin but not reactive blue 2 in blocking this pathway further indicate the involvement of the P_2X1 purinoceptor subtype in activation of lymphatic smooth muscle by ATP. P_2X1 receptors are known to be expressed at high levels in the smooth muscle cells of peripheral arteries (Evans & Surprenant, 1992; Vulchanova et al., 1996) and can be blocked by PPADS and suramin but not reactive blue 2 (Ralevic & Burnstock, 1998). In addition, all other receptor subtypes except for P_2X3, which are expressed in peripheral sensory ganglia, have been found to have low affinities to ATP and are insensitive to α,β-MeATP (Collo et al., 1996; Vulchanova et al., 1996). P_2X4 and P_2X5 have also been reported in smooth muscle (Gitterman & Evans, 2000; Lewis & Evans, 2000a). However they are unlikely to be involved in lymphatic vasomotion as they are relatively insensitive to α,β-MeATP and the P_2X4 receptor isoform is suramin and PPADS-insensitive (Gitterman & Evans, 2000; Lewis & Evans, 2000a). A distinguishing feature of P_2X3 receptors, which suggests they do not play a role in ATP activation of lymphatic smooth muscle, is that the receptors do not readily desensitize (Ralevic & Burnstock, 1998). Surprenant et al. (2000) have reported P_2X(1/5) heterometric receptors in guinea-pig submucosal arteries. However it is unlikely that these receptors are involved in ATP-induced response, as these receptors exhibit a very high sensitivity to ATP (e.g. responses obtained at 3 nM) and have currents that do not desensitize. In contrast, our present experiments have shown that only high [ATP] (>1 μM) produced significant increases in lymphatic smooth muscle [Ca²⁺]_i which returned to baseline within 5 min.

The finding that ATP-induced increases in lymphatic smooth muscle [Ca²⁺]_i and vasomotion in vessels in which P_2X receptors had been desensitized by α,β-MeATP suggests a role for P_2Y receptors in these responses. In addition, responses similar to those induced by ATP were activated by UTP, an agonist of P_2Y2, P_2Y4 and P_2Y6 purinoceptors. P_2Y2, P_2Y4 and P_2Y6 purinoceptors have been reported to be present in vascular smooth muscle and there is functional evidence to suggest that they can modulate vasoconstriction (Erlinge et al., 1998; Ralevic & Burnstock, 1998). P_2Y receptors can be differentiated from each other by their pharmacological properties. UTP has been found to be an agonist of suramin-sensitive P_2Y2 receptors in rat (Boarder & Hourani, 1998) and suramin-insensitive P_2Y4 receptors. In contrast, P_2Y6 receptors are UDP selective and insensitive to suramin (Webb et al., 1998). Therefore, on the basis of information presently available for this tissue, P_2Y2 are the most likely receptor subtype involved in smooth muscle response to ATP. A possible involvement of P_2Y1 receptors is unlikely as this receptor sub-type is sensitive to ATP but rather insensitive to UTP. In contrast, lymphatic P_2Y receptors were found to be equally sensitive to ATP and UTP.

Our previous experiments have shown that the P_2Y receptors in lymphatic endothelium exert their role through activation of a PTx-sensitive G protein (Gao et al., 1999). In contrast, our present results show that P_2Y receptors in lymphatic smooth muscle are PTx-insensitive, indicating the involvement of a different G protein.

The ATP-induced increases in mesenteric lymphatic smooth muscle [Ca²⁺]_i were found to be biphasic, exhibiting an initial transient followed by a sustained phase where [Ca²⁺]_i remained elevated. Such biphasic responses to ATP have been observed in a variety of tissues and have been reported to be due to a combination of internal Ca²⁺ release and Ca²⁺ influx. The initial transient of the ATP-induced increase in [Ca²⁺]_i is likely to be due to IP₃-modulated Ca²⁺ release from the SR as the response was blocked by the PLC inhibitor U73312, by the IP₃ receptor blocker heparin and by the store Ca²⁺ ATPase inhibitor CPA. Similar findings have been reported in vascular smooth muscle (el-moatassim et al., 1992). The sustained component of the ATP induced increase in [Ca²⁺]_i was found to be dependent on extracellular Ca²⁺ influx and resulted from P_2X-gated influx with a second pathway associated with functional Ca²⁺ stores. Evidence for this was provided by the finding that the sustained phase of ATP-induced increase in [Ca²⁺]_i was absent when external solutions were effectively free of Ca²⁺. Part of this response arose through influx of Ca²⁺ through P_2X-gated channels as ATP caused a sustained increase in smooth muscle [Ca²⁺]_i after depletion of the intracellular stores with CPA, although the sustained increase was now significantly smaller. However, the combination of CPA-induced store depletion and desensitisation of P_2X receptors abolished the slow phase of the ATP response (Figure 9). Evidence for a sustained source of Ca²⁺ entry second to that resulting from P_2X receptors was also provided by the finding that ATP-induced increases in smooth muscle [Ca²⁺]_i in vessels in which P_2X receptors had been desensitized caused a transient and sustained response (Figure 5c). A likely candidate for this store-associated Ca²⁺ entry is SOCs.

The physiological concentration of ATP in human plasma is normally 0.1–1 μM (Forrester & Lind, 1969; Ryan et al., 1996). At these levels ATP would modulate lymphatic vasomotion by stimulating the release of both endothelium-derived relaxing and constrictive factors, though predominantly the l atter (Gao et al., 1999). However, under some pathophysiological conditions (e.g inflammation and shock), the concentration of ATP may increase and reach much higher levels (e.g 0.1 mM) (Gordon, 1986). Such conditions may be particularly relevant to lymphatic function. In conclusion, our data indicates that ATP exerts its effects on smooth muscle by activating both P_2X1 and P_2Y2 purinoceptors. The resultant increases in smooth muscle [Ca²⁺]_i, tone and vasomotion are likely to be due to P_2Y2 receptor-mediated Ca²⁺ release from the SR and Ca²⁺ influx from the extracellular space by P_2X1-gated channels and SOCs.

Abbreviations

ACh

acetylcholine

α,β-MeATP

α,β-Methylene ATP

ATP

adenosine 5′-triphosphate

CPA

cyclopiazonic acid

IP₃

inositol-1,4,5-trisphosphate

PLC

Phospholipase C

PPADS

pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid

SNP

sodium nitroprusside

SOCs

store-operated channels

UTP

uridine 5′-triphosphate