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1Mucosal Inflammation and Smooth Muscle Research Groups, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Alberta, Canada T2N 4N1; and 2Neuroscience Group, School of Biomedical Sciences, Faculty of Health, University of Newcastle, Callaghan, New South Wales, Australia 2308
Submitted 4 August 2003 ; accepted in final form 26 January 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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pumping; nitric oxide; smooth muscle; lymphatic vasomotion
CPA, a mycotoxin from Aspergillus and Penicillium, has been described as a highly selective inhibitor of Ca2+-ATPase in skeletal, cardiac, and smooth muscle SR (13, 52). In the latter tissue, CPA has been shown to reversibly inhibit ATP-dependent Ca2+ uptake of the SR in skinned ileal smooth muscle cells (45) and is proposed to do so in intact smooth muscle preparations of the aorta (8), trachea (6), and mesenteric artery (26). In addition, CPA has been reported to decrease lymphatic pumping in isolated bovine mesenteric lymphatic vessels (3). The present study presents a detailed characterization of the actions of CPA. It is found that CPA acts by two primary pathways to decrease lymphatic pacemaking and underlying STD activity.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Small collecting lymphatic vessels (diameter, 80–230 µm) that supply the jejunum and ileum were dissected together with their associated artery and vein and were left intact within the surrounding mesentery. The mesentery was used to pin out the tissue onto the Sylgard-coated bottom of a small organ bath (volume, 100 µl), and the preparation was mounted on the stage of an inverted microscope (TMS; Nikon). The pinning procedure appeared to exert only mild physical forces on the lymphatics; it tended to flatten the vessels but caused little if any stretch due to the vessels running inside pocketlike regions of the mesentery such that the mesentery absorbed the stretch. Such unperfused vessels showed relatively little spontaneous activity under resting conditions but could readily be stimulated by vessel perfusion or application of excitatory agonists. Experiments were performed with tissues continuously superfused with PSS heated to 36°C at a flow rate of 3 ml/min, which caused 90% changeover in <7 s.
Electrophysiology.
Resting membrane potential was measured using conventional glass intracellular microelectrodes with resistances of 150–250 M
when filled with 0.5 M KCl. Electrodes were connected to an amplifier (Intra 767; World Precision Instruments; Sarasota, FL) through an Ag-AgCl half cell. Resting membrane potential was monitored on a digital oscilloscope (VC6525; Hitachi) and simultaneously recorded on a computer via an analog-to-digital converter (PowerLab/4SP, AD Instrument; Mountain View, CA). Intracellular recordings were obtained by impaling smooth muscle cells from the adventitial side of the vessels. Vessels were cut into short segments (125–350 µm) with fine dissecting scissors to ensure simplified electrical properties of the smooth muscle. In this situation, electrical activity, although generated at localized foci within the smooth muscle, produces a similar potential change in all the smooth muscle cells of the segment (48). Endothelial membrane potential recordings were obtained either by deeper penetration of the microelectrode into the vessel or by impaling the first layer of cells in a vessel that has been carefully cut open with the intima then facing up. As described in a previous study (56), endothelial cells are more polarized than smooth muscle cells (resting membrane potential, 
72 vs. 55 mV) and have a characteristically different response to ACh. These two criteria were used to discriminate between endothelial and smooth muscle intracellular recordings.
Lymphatic smooth muscle impalements were characterized by a sharp drop in potential that settled after 10–15 s to a value typically more negative than –45 mV. Impalements were maintained for >5 min in >90% of the cases and 
3 h optimally. CPA was added to the PSS that superfused the preparation either for short (1 min) or longer (4–10 min) durations. In experiments where the effects of CPA were assessed in the presence of inhibitors {NG-nitro-L-arginine (L-NNA), 1H-[1,2,4]oxadiazolol-[4,3-a]quinoxaline-1-one (ODQ), and glibenclamide}, CPA was applied first as a control and then at least 20 min later in the presence of the antagonist, which had been superfused for at least 10 min. This protocol was usually performed during the same impalement. However, in some instances, successive impalements were obtained from neighboring cells in the same segment. There was no significant difference in the responses for repeated treatments of CPA applied at the same concentration 20 min apart.
STD activity was assessed by measuring the frequency and amplitude of events >1 mV in size. STD frequency and amplitude were measured for 15–60 s (depending on the stability of the recording, but typically 30 s) before application of CPA (or other agonists: U-46619, norepinephrine, thimerosal) and were then compared with those occurring for a period of the same duration during the time period when response to the agonist was maximal.
Functional removal of endothelium.
The lymphatic endothelium was destroyed in vitro after a procedure that has been previously described (11, 12). Briefly, a fine glass micropipette was inserted into the lumen of a previously cut vessel. The micropipette, which was connected to an infusion pump via Teflon tubing, was used to luminally perfuse the vessel with PSS in the direction of the valves. This procedure induced rhythmic constrictions of the vessel. To functionally destroy the endothelium, small air bubbles were passed in repeated streams (5–6 times for 5–10 s; rate, 3–5 µl/min) through the vessel lumen via this micropipette. The success of the endothelial destruction was confirmed by applying ACh (10 µM) before sodium nitroprusside (100 µM) in the superfusion solution while the vessel lumen was perfused. An absence of an ACh-induced decrease in contractions as is observed in endothelium-intact vessels and a decrease in contractions to sodium nitroprusside were used as confirmation of the success of the procedure. Endothelial destruction based on this testing procedure proved successful in 50% of treated vessels. The use of sodium nitroprusside was necessary, as it has been shown that 40% of guinea pig mesenteric lymphatic vessels with intact endothelium exhibit a high basal production of NO and hence do not respond in any way to either ACh or sodium nitroprusside (54). Loss of function of the endothelium was further confirmed during the electrophysiological experiments by the absence of endothelium-derived hyperpolarization and an inability to significantly change STD activity in response to 10 µM ACh. Membrane potential responses to ACh are very reliable, as they occur in >95% of the cells with functional endothelium.
Ratiometric measurement of [Ca2+]i. Experiments involving measurement of [Ca2+]i changes in the lymphatic smooth muscle or endothelium were performed using the calcium-sensing fluorescent dye fura-2. The smooth muscle of lymphatic vessels was loaded by luminally perfusing endothelium-denuded vessels at room temperature with the membrane permeant fura-2 acetoxymethyl ester (AM, 2 µM) and pluronic acid (0.2% wt/vol) for 30 min. The same procedure was used in endothelium-intact vessels to specifically load the endothelium. After this, the vessel was perfused with control solution for 10 min to wash out extraneous dye. The vessel was then left for an equilibration time of at least 20 min to allow the intracellular esterases to cleave fura-2 AM (14). The mesentery was hooked on small pins glued to the outside of a metal frame (1 x 1 cm) such that the frame positioned the mesentery and associated lymphatic vessels flat against the glass coverslip that formed the bottom of the organ bath (volume, 0.5 ml). The tissue was superfused with heated (34–36°C) PSS at a rate of 6 ml/min, and regions of individual vessel chambers (lymphangions) were viewed by an inverted microscope (Zeiss Axiovert 10) using a x40 oil-immersion objective (numerical aperture, 1.3). Ratiometric experiments were made photometrically on vessel chambers. Each such measurement incorporated the response from multiple endothelial or smooth muscle cells in endothelium-intact or -denuded vessels, respectively.
Ratiometric fura-2 measurements were made with vessel chambers alternatively illuminated by a xenon lamp at 340- and 380-nm wavelengths for durations of 50 ms for each wavelength with a 50-ms interval between each exposure. This cycle was repeated at a frequency of 5 Hz. Emission light was passed through a dichroic mirror (490 nm) and a band-pass filter (510 nm) and collected by a photomultiplier; the output response was digitized and captured by computer. Experiments were performed in the absence of luminal perfusion to minimize movement. These movements were then sufficiently small to allow the fura-2-loaded smooth muscle or endothelium to remain in the focal plane. Such movement could in some cases cause the fluorescence at the 380-nm wavelength (F380) to increase instead of decrease. This most likely occurred due to more dye-loaded tissue moving into focus and causing a net increase in F380 fluorescence. This is accounted for by a proportional change in the signal for fluorescence at the 340-nm wavelength (F340) so that the ratio cancels out such movement artifacts if the ratios are obtained at a time interval of sufficiently short duration during which movement is small. Independent measurement of constrictions suggests that this was upheld during most of the constriction except for the period of maximum onset when there was some error. Vessel chambers typically constricted to peak amplitude in <0.5 s and then decayed relatively slowly to baseline over the next 2–3 s. The protocol used to measure the F340/F380 ratios incurred a maximum interval between ratios of 100 ms during which there could be up to 25% movement during the maximum-onset phase of constriction but less than 5% during the decay phase. Thus the ratios would only be substantially distorted during the maximum-onset phase of constriction (i.e., during the first 0.5 s of the Ca2+ transient).
Chemicals and drugs. ACh, CPA, glibenclamide, L-NNA, norepinephrine, sodium nitroprusside, and thimerosal were all purchased from Sigma; fura-2 AM was from Molecular Probes (Eugene, OR), ODQ was from Alexis (San Diego, CA), and U-46619 was from Cayman Chemicals (Ann Arbor, MI). CPA, fura-2 AM, glibenclamide, ODQ, and U-46619 were dissolved in dimethylsulfoxide; L-NNA was dissolved in 0.1 M HCl, and the remainder were dissolved in distilled water to yield 10 mM stock solutions. After dilution of the drugs to their final concentrations in physiological saline, the diluted vehicle had no effects on tissue activity.
Data analysis. Experimental data are expressed as means ± SE. Statistical significance was assessed using a two-tailed, paired Student's t-test (unless specified otherwise in the text) with P < 0.05 being considered significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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95% of the lymphatic smooth muscle recordings (see Refs. 48, 54) was also reduced in a concentration-dependent manner (Fig. 1, C and D). At the peak of the hyperpolarization induced by CPA (10 µM), STD frequency was significantly reduced to 48 ± 7% of control and STD amplitude declined to 76 ± 4% of control (n = 21; P < 0.01). In five recordings, the CPA-induced hyperpolarization was preceded by a small depolarization of 1–4 mV and an associated increase in STD frequency and amplitude (143 ± 24 and 134 ± 11% of control, respectively). Alterations in STD activity and hyperpolarization persisted for 3–5 min after the CPA had been washed out before returning to control values.
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Endothelium-independent effects of CPA on STDs. To avoid the confounding involvement of EDNO, the effects of 10 µM CPA on smooth muscle STD activity were investigated either on endothelium-denuded vessels or in the presence of 100 µM L-NNA or 10 µM ODQ. As illustrated in Fig. 2A, in endothelium-denuded vessels, lymphatic smooth muscle responded to 10 µM CPA with an initial and transient increase in STD activity that was associated with a small depolarization (108 ± 7 and 115 ± 11% of control for frequency and amplitude, respectively; n = 4) that sometimes culminated in action potentials. This phase, which lasted for 1–2 min, was then followed by a marked and prolonged CPA-induced decrease in STD activity. STD frequency and amplitude were reduced to 42 ± 13 and to 77 ± 4% of control, respectively (n = 6; Fig. 2D). Similar findings were identified in the presence of L-NNA or ODQ (10 µM) with respective CPA-induced decreases in STD frequency and amplitude of 32 ± 6 and 62 ± 7% of control for L-NNA (n = 9; Fig. 2D) and 52 ± 11 and 70 ± 5% of control for ODQ (n = 6). These values were in all cases not significantly different from the responses induced by 10 µM CPA on smooth muscle in vessels with functional endothelium (P > 0.1 for all cases, unpaired Student's t-test). Upon washout of the CPA, the membrane potential repolarized to control values, sometimes with a transient hyperpolarization, whereas STD activity returned to control levels within 3–5 min.
Endothelium-independent effects of CPA on agonist-evoked STDs.
The effects of CPA were also investigated during agonist-induced enhancement of STD activity. As described in the previous section, these experiments were performed under conditions where the EDNO contribution was minimized. Experiments were first performed using the thromboxane A2 mimetic U-46619. At a concentration of 0.1 µM, U-46619 increased STD frequency and amplitude (n = 6; Fig. 3, A and D; see also Ref. 57). U-46619 (0.25 µM) caused increased STD activity, a large transient depolarization, and the near-rhythmic generation of action potentials (Fig. 4A). The U-46619-induced increase in STD activity was significantly reduced below control levels (P < 0.05) during application of 10 µM CPA to vessel segments (n = 6; Fig. 3, A and D). Experiments were also performed in the presence of norepinephrine, which enhances STD activity through activation of 
-adrenoceptors (Fig. 3D; Ref. 48). Application of 10 µM CPA to vessel segments exposed to 10–50 nM norepinephrine reduced STD frequency and amplitude (n = 4; P < 0.05; Fig. 3, B and D). The third activator used was thimerosal, an agent reported to sensitize inositol 1,4,5-trisphosphate (IP3) receptors and lead to enhancement of IP3-induced Ca2+ release (4, 5, 18). Application of 1 µM thimerosal increased STD frequency and amplitude to 128 ± 12 and 148 ± 35% of control, respectively (n = 5). Application of 10 µM CPA to vessel segments exposed to 1 µM thimerosal reduced STD frequency and amplitude to values below control (n = 5; Fig. 3, C and D). The membrane potential usually depolarized by 2–5 mV in the presence of any of these agonists and by another 4–10 mV upon CPA application.
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These effects closely compare with the action of CPA on the smooth muscle membrane potential shown in Fig. 4A. In this preparation, U-46619 was added at a concentration that increased STD activity and caused a transient depolarization of the membrane potential and near-rhythmic generation of action potentials. Application of CPA (10 µM) in the continued presence of U-46619 caused a gradual depolarization during which there was an initial increase in the frequency of action potentials and subsequent inhibition of these events (Fig. 4A).
Direct action of CPA on endothelium. The effects of CPA on the endothelium were investigated by measuring changes in both [Ca2+]i and membrane potential. CPA (10 µM) caused [Ca2+]i to increase to 123 ± 2% of control (n = 3), which is a value similar to that obtained in the same preparations with 10 µM ACh (122 ± 5%). However, the kinetics of the onset of the increase in [Ca2+]i occurred some 10-fold slower for CPA than for ACh with the times for the response to increase to 50% of peak amplitude 103 ± 7 and 10 ± 1 s, respectively (Fig. 5A).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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CPA caused the lymphatic smooth muscle membrane potential to hyperpolarize and the STD frequency and amplitude to decrease. The hyperpolarization was shown to be primarily mediated by the CPA-induced production and release of NO from the lymphatic endothelium, as it was strongly reduced in the presence of L-NNA and ODQ as well as in vessels with nonfunctional endothelium. Inhibition of CPA-induced hyperpolarization by glibenclamide further suggests an involvement of EDNO, as NO has been shown to activate ATP-sensitive K+ channels in the same lymphatic vessels (53). Although NO itself has been shown to reduce STD activity (57), we demonstrate here that the CPA-induced decrease in STD activity was largely independent of EDNO, as CPA still markedly reduced STD activity while endothelial function or NO production was impaired. Consistent with results obtained from other smooth muscles (15, 16, 50), our findings suggest that CPA acts directly on lymphatic smooth muscle to inhibit STD generation. At the concentration range used in the present study (1–10 µM), CPA has been reported to selectively alter SR Ca2+-ATPase function in smooth muscle (see Refs. 6, 45). The decrease in lymphatic smooth muscle STD activity is thus likely to result from the action of CPA on Ca2+-ATPase, and it correlates with the expected effect of CPA on intracellular Ca2+ handling. Moreover, the small depolarization and initial increase in STD activity that were observed early during CPA superfusion may be explained by the transient increase in [Ca2+]i, which is thought to occur as a first event before store depletion, when the reuptake of Ca2+ into the SR is altered (21, 41, 58). Consistent with this, we found that CPA increased [Ca2+]i (see also Ref. 60). The actions of CPA on pacemaker events were also assessed in the presence of norepinephrine and the thromboxane A2 mimetic U-46619. These agonists have been shown to increase lymphatic pacemaker activity (12, 20, 30, 39, 48, 57) most likely through increased production of IP3 (9, 35). Pacemaker events (i.e., large STDs or summations thereof) are interpreted to reflect synchronized release of Ca2+ from localized groups of IP3 receptor-operated stores: the Ca2+ release generates inward current through activation of pathways such as Ca2+-activated Cl– channels (see Ref; 22). Recent studies on pacemaking in another smooth muscle (guinea pig gastric pylorus) indicate that IP3-mobilizing agonists induce pacemaking by enhancing the number of active IP3 receptor-operated stores, and these are then entrained by a coupled oscillator-based mechanism to generate large-amplitude pacemaker potentials (49). This interpretation fits with the known actions of norepinephrine and U-46619 to enhance lymphatic vasomotion and underlying STD activity. It also fits with the finding of similar actions by the thiol-oxidizing reagent thimerosal, which at the concentration used (1 µM) is known to selectively stimulate Ca2+ release by sensitizing IP3 and/or ryanodine receptors (dependent on the tissue; Refs. 1, 4, 5, 18, 38, 43). Importantly, CPA actions to reduce Ca2+-release events measured by recording STDs are also consistent with this model, as stores would be depleted by CPA-induced block of the store Ca2+ ATPase (21, 41). However, the finding that there was residual STD activity in the absence or presence of the agonists indicates that CPA-induced inhibition of store function is incomplete for the range of CPA concentrations tested or that additional sources of Ca2+ are available for STDs to occur.
Investigations of CPA action on the lymphatic endothelium revealed an increase in endothelial [Ca2+]i upon CPA stimulation. Although observed here for the first time in lymphatic endothelium, CPA-induced increases in endothelial [Ca2+]i are well documented in many vascular preparations (17, 24, 25). The increase in endothelial [Ca2+]i caused by CPA, like that caused by many other agonists, has been shown to be necessary to the release of endothelium-derived substances such as EDNO (19, 33, 61). The lymphatic endothelium plays an important role in modulating lymphatic pumping (10, 37, 59). In particular, the lymphatic endothelium was shown to slow lymphatic pumping by releasing EDNO both in vitro (32, 40, 54, 59) and in vivo (42). An increase in endothelial [Ca2+]i, which is associated with the production and release of EDNO, has also been demonstrated in lymphatic vessels of the guinea pig mesentery in response to ACh (54). This ACh-induced action is accompanied by marked depolarization of the lymphatic endothelium (56). These findings are consistent with the present observations that CPA increases lymphatic endothelial [Ca2+]i and depolarizes membrane potential. However, the rates for both of these changes were much slower than those observed in response to ACh, which is consistent with CPA acting through recruitment of different mechanisms. A gradual increase in [Ca2+]i with a similar time course has been reported for the CPA-induced response of rabbit valvular endothelial cells (25). Although membrane potential was not measured in the latter study, a CPA-induced increase in [Ca2+]i in rabbit aortic valvular endothelial cells has been associated with hyperpolarization of endothelium (36). Hyperpolarizations to vasoactive agonists have been described for many endothelial cells and are proposed to provide the electrochemical driving force for Ca2+ entry into endothelial cells (see Ref. 23) thus promoting the Ca2+-dependent production of EDNO (27). The finding that the lymphatic endothelium underwent depolarization in response to CPA and ACh does not fit this logic, but such responses have also been reported for some vascular endothelial cells in response to vasoactive substances or Ca2+-ATPase inhibitors (29, 31, 47).
In conclusion, responses of lymphatic smooth muscle to CPA are suggested to be mediated by two mechanisms: a direct action of CPA through depletion of smooth muscle Ca2+ stores, which inhibits the activity of pacemaker-associated Ca2+ release events measured as STDs, and an action of CPA on endothelium to release EDNO, which causes smooth muscle to hyperpolarize and decrease STD activity. Both actions are proposed to contribute to a decrease in lymphatic pacemaking and the resultant propulsion of lymph.
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FOOTNOTES |
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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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REFERENCES |
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-Adrenergic inhibition of bovine mesenteric lymphatics. J Physiol 374: 401–411, 1986.-adrenoceptor agonist-induced hyperpolarizations. Br J Pharmacol 125: 17–22, 1998.[CrossRef][ISI][Medline]
-Adrenoceptor-mediated hyperpolarization in lymphatic smooth muscle of guinea pig mesentery. Am J Physiol Heart Circ Physiol 270: H1687–H1695, 1996.This article has been cited by other articles:
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  M. S. Imtiaz, J. Zhao, K. Hosaka, P.-Y. von der Weid, M. Crowe, and D. F. van Helden Pacemaking through Ca2+ Stores Interacting as Coupled Oscillators via Membrane Depolarization Biophys. J., June 1, 2007; 92(11): 3843 - 3861. [Abstract] [Full Text] [PDF]
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see also A, bottom) and during inhibition by CPA (
) are compared on an expanded time scale. D: concentration-dependent relationship of the decreases in frequency and amplitude of STDs measured at the peak of CPA-induced hyperpolarization expressed as a percentage of the values obtained during the same impalement before CPA application (n = 5–21). Columns are means ± SE. *P < 0.05 vs. control (paired Student's t-test). Resting membrane potential values in this and subsequent figures are indicated left of traces. [CPA], CPA concentration.
