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Am J Physiol Regul Integr Comp Physiol 294: R1524-R1532, 2008. First published February 27, 2008; doi:10.1152/ajpregu.00888.2007
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NEUROHUMORAL CONTROL OF CARDIOVASCULAR FUNCTION

Calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics

Patrick J. Dougherty,1 Michael J. Davis,2 David C. Zawieja,1 and Mariappan Muthuchamy1

1Department of Systems Biology and Translational Medicine, Cardiovascular Research Institute, Division of Lymphatic Biology, Texas A&M Health Science Center College of Medicine, College Station, Texas; and 2Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, Missouri

Submitted 12 December 2007 ; accepted in final form 26 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lymphatic muscle contraction is critical for the centripetal movement of lymph that regulates fluid balance, protein homeostasis, lipid absorption, and immune function. We have demonstrated that lymphatic muscle has both smooth and striated muscle contractile elements; however, the basic contractile properties of this tissue remain poorly defined. We hypothesized that contractile characteristics of lymphatic myofilaments would be different from vascular smooth muscle myofilaments. To test this hypothesis, –log[Ca2+] (pCa)-tension relationship was determined for {alpha}-toxin permeabilized mesenteric lymphatics, arteries, and veins. The Ca2+ sensitivity (pCa50) of mesenteric lymphatics was significantly lower compared with arteries (6.16 ± 0.05 vs. 6.44 ± 0.02; P < 0.05), whereas there was no difference in pCa50 between lymphatics and veins (6.16 ± 0.05 vs. 6.00 ± 0.10; not significant). The Hill coefficient for {alpha}-toxin-permeabilized lymphatics was not significantly different from arteries but was significantly greater than that of the veins (1.98 ± 0.19 vs. 1.21 ± 0.18; P < 0.05). In addition, the maximal tension and pCa50 values were significantly greater in {alpha}-toxin-permeabilized lymphatics compared with β-escin-permeabilized lymphatics (0.27 ± 0.03 vs. 0.15 ± 0.01 and 6.16 ± 0.05 vs. 5.86 ± 0.06 mN/mm, respectively; P < 0.05), whereas the Hill coefficient was significantly greater in β-escin-permeabilized lymphatics. Western blot analyses revealed that CPI-17 levels were significantly decreased by about 50% in β-escin-permeabilized lymphatics, compared with controls, whereas no change in the level of calmodulin was detected. Our data constitute the first description of the pCa-tension relationship in permeabilized lymphatic muscle. It suggests that differences in myofilament Ca2+ sensitivity and cooperativity among lymphatic muscle and vascular smooth muscles contribute to the functional differences that exist between these tissues.

lymphatic muscle; pCa-force relationship; calcium sensitivity

THE LYMPHATIC VASCULAR SYSTEM plays an important role in fluid and protein homeostasis, lipid absorption, and immune function (47). Lymphatic transport of fluids, macromolecules, and immune cells from the peripheral tissues to the blood vascular system occurs against a net pressure gradient. Thus, lymph transport relies on a system of lymph pumps and valves to drive and direct lymph flow. Impairment of the lymph pump has been documented in several pathological conditions, such as aging (13), inflammatory bowel disease (45), and in women following lymph node resection to treat breast cancer (32), and is associated with lymphedema and an increased risk of infection in the affected region (28, 36). Despite its critical role in health and disease, relatively little is known about basic contractile properties of lymphatic muscle (47).

Extrinsic and intrinsic mechanisms contribute to the movement of lymph through the lymphatic system. Initial lymphatics do not possess muscle cells and rely upon extrinsic factors, such as mechanical compression from skeletal muscle, to move lymph downstream into collecting lymphatics, which are lined with one or more layers of muscle cells. Collecting lymphatics possess a patent one-way valve system that promotes unidirectional flow and divides the collecting vessels into smaller functional units, termed lymphangions. The intrinsic lymph pump refers to the strong, brisk phasic contractions of lymphatic muscle that serve to propel lymph into downstream lymphangions, while the patent one-way valves prevent retrograde lymph flow. The phasic contractions of lymphatic muscle are initiated by a depolarizing action potential and are associated with transient calcium spikes. Because collecting lymphatics exhibit distinct cycles of systole and diastole, the lymph pump is often analyzed using cardiac function indices (3).

Lymphatic muscle is a highly specialized type of smooth muscle that exhibits both tonic and phasic contractions that are responsible for the distinct periods of diastole and systole, respectively, observed in collecting lymphatics. Both tonic and phasic contractions are sensitive to changes in pressure and flow (11, 12), extracellular Ca2+ concentration (29), and agonist stimulation (1, 2). The phasic contractions in rat mesenteric lymphatics occur at frequencies of ~5–15 contractions per minute (48). Interestingly, the shortening velocity of lymphatic muscle has been reported to be ~2- and ~5-fold faster than that of phasic and tonic smooth muscle, respectively, and closer to that of striated muscle (3). In support of the unique functional behavior of lymphatic muscle, we have recently reported that the lymphatic muscle contractile protein (i.e., actin and myosin) expression profile is composed of both cardiac/skeletal and smooth muscle isoforms (33). Our results demonstrated that rat mesenteric lymphatics express only smooth muscle B (SMB) smooth muscle myosin heavy chain (SM-MHC), whereas, nearby mesenteric arterioles expressed both smooth muscle A (SMA) and SMB isoforms. In addition, slow-skeletal/fetal cardiac muscle-specific β-MHC message was detected only in mesenteric lymphatics. Furthermore, all four actin messages, {alpha}-cardiac, {alpha}-vascular, {gamma}-enteric, and {alpha}-skeletal, were present in both mesenteric lymphatics and arterioles. Western blot and immunohistochemical analyses corroborated the mRNA studies with the exception that only {alpha}-vascular actin protein was detected in arterioles. This combination of smooth and striated muscle contractile elements present in lymphatic muscle could provide unique contractile characteristics (i.e., Ca2+ sensitivity and cooperativity) for lymphatic myofilaments. Although numerous mechanical (3, 12, 30, 35, 49) and neurohumoral (1, 2, 19, 31, 46, 50) factors have been shown to modulate the tonic and phasic activity of collecting lymphatics, few investigations of the basic contractile properties of the lymphatic myofilament have been published.

Smooth muscle is generally classified as either tonic or phasic based on its contractile behavior (38). Regulation of smooth muscle contraction occurs primarily through the phosphorylation of the 20-kDa regulatory myosin light chain (MLC20), which is sensitive to the relative activities of MLC kinase (MLCK) and MLC phosphatase (MLCP) (37). A number of published reports have indicated that significant differences exist between the molecular composition and the contractility of myofilaments from tonic and phasic smooth muscle. For example, phasic smooth muscle expresses higher levels of the acidic 17-kDa myosin light chain isoform (39), the MLCP regulatory subunit, MYPT1, (44), and the thin-filament proteins caldesmon and calmodulin (40). Reports indicate that both MLCK and MLCP activities are higher in phasic compared with tonic smooth muscle (15, 40). On the other hand, tonic smooth muscle expresses higher levels of the calcium-sensitizing protein, CPI-17 (protein kinase C-potentiated inhibitor protein of 17 kDa), which is a potent inhibitor of MLCP when activated via phosphorylation of Thr38 (17). In addition to the molecular differences between tonic and phasic smooth muscle myofilaments, tonic vascular smooth muscle treated with the mild permeabilizing agent, {alpha}-toxin, has been shown to exhibit significantly greater Ca2+ sensitivity than does phasic vascular and visceral smooth muscle (24). Although detailed comparisons have been made between the myofilaments from tonic and phasic smooth muscle from different organ systems, no such data are available for lymphatic muscle, which exhibits both tonic and phasic activities.

In recent studies characterizing the mechanical properties of lymphatic muscle, we have shown that mesenteric lymphatic muscle exhibits striking contractile differences when compared with small arteries and veins from the same vascular bed. Though lymphatic vessels generate the least active tension and stress among these three vessels, the lymphatic vessels exhibited distinct elastic characteristics, as well as both phasic and tonic contractions (4, 48, 49). Because tonic and phasic smooth muscle myofilaments exhibit different biochemical characteristics and the lymphatic muscle has unique biomechanical properties, we hypothesized that the calcium sensitivity and cooperativity of lymphatic myofilaments must be different from tonic vascular smooth muscle to attain its unique phasic and tonic contractile nature. To test this hypothesis, we determined the calcium-tension relationship in permeabilized mesenteric lymphatic segments and compared it with the calcium-tension relationships of permeabilized mesenteric arteries and veins.

The use of permeabilized smooth muscle preparations provides important information regarding basic biochemical properties, such as calcium sensitivity (pCa50) and cooperativity (nH) of contraction at the myofilament level. There are several methods frequently used to permeabilize or skin smooth muscle; these include {alpha}-toxin, β-escin, saponin, and Triton X-100. Treatment with {alpha}-toxin or β-escin results in permeabilized membranes that maintain receptor-coupled signaling pathways (25, 27) that are disrupted after treatment with saponin or Triton X-100. For this reason, {alpha}-toxin- or β-escin-permeabilized preparations have been critical in determining the complex interactions between agonist stimulation and myofilament activation in smooth muscle. However, the size of the pores created by {alpha}-toxin and β-escin differs substantially. Permeabilization with {alpha}-toxin results in the formation of small transmembrane pores that allow the passage of ions and small molecules but limits the movement of large molecules (8), whereas β-escin creates larger pores that allow passage of ions and small and larger molecules across the permeabilized membrane (21). Because of the advantages and disadvantages inherent in the different permeabilization methods and the conflicting reports regarding the effects of permeabilization treatment on smooth muscle contractility (41, 42), we also determined the effects of two types of permeabilization ({alpha}-toxin and β-escin) on the calcium-tension relationship of permeabilized lymphatic muscle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and procedures. Male Sprague-Dawley rats (250–400 g) were anesthetized with intramuscular injections of diazepam (2.5 mg/kg body wt) and a fentanyl/droperidol solution (0.3 ml/kg). A midline incision was made through the skin, fascia, and underlying abdominal muscle layers, and a 6- to 7-cm loop of the small intestine was exteriorized. With the assistance of a stereomicroscope (Olympus SZX12, Leeds Instruments, Irving, TX), a suitable mesenteric artery, vein, or lymphatic was identified and carefully cleaned of fat and connective tissue, while being continuously superfused with PBS. A 1-cm-long segment of the vessel to be studied was dissected free and transferred to a 35-mm Petri dish containing PBS. After the tissue was removed, the animal was killed by cervical dislocation. All animal procedures were reviewed and approved by the Texas A&M University Laboratory Animal Care Committee and adhered to both institutional and federal guidelines.

Isolated vessel procedures. Two stainless steel wires (40 µm OD for arteries and veins; 25 µm OD for lymphatics) were passed, one at a time, through the lumen of a 2-mm segment of the isolated mesenteric vessel. The vessel was then transferred to the organ chamber of a single-channel, wire myograph (Model 310A, Danish Myo Technology, Aarhus, Denmark) that contained a physiological salt solution (PSS) of the following composition (in mM): 145.0 NaCl, 4.7 KCl, 2 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 sodium pyruvate, 0.02 EDTA and 3.0 3-(N-morpholino) propanesulfonic acid (MOPS). One wire was secured to the jaw of the myograph that was under control of an adjustable micrometer, which was used to stretch the vessel between the two parallel wires. The other wire was secured to the other jaw of the myograph coupled to a calibrated force transducer (Danish Myo Technology). The force transducer measured forces between 0 and 30 mN and the force output was digitized with a PCI-6030e A–D card and interface (National Instruments, Austin, TX). Experiments were recorded using LabView (National Instruments) at a sampling frequency of 10 Hz and stored on a PC (Dell, Austin, TX). Once the vessel was successfully mounted, the myograph was transferred to the stage of an inverted microscope (Olympus CKX41, Leeds Instruments, Irving, TX) coupled to a CCD camera (Model PK-M2U, Hitachi Denshi, Woodbury, NY). A video micrometer (Microcirculation Research Institute, Texas A&M University, College Station, TX) was used to manually measure inner diameter.

Length-tension protocol. A vessel segment was mounted in the myograph, and the bathing solution was maintained at 25°C. The experimental protocols were initiated after a 30- to 60-min equilibration period during which the bathing solution was changed once. We first attested the length-activated tension relationship of each vessel, as described previously (49). Briefly, vessels were stretched to a range of predetermined preloads. After a steady preload was achieved, vessels were maximally activated with KPSS (PSS with equimolar substitution of KCl for NaCl). For the experiments with mesenteric lymphatics and veins, the KPSS was supplemented with 1 µM Substance P (SP; KPSS+SP). The optimal circumferential length (L0) was defined as the internal circumference that resulted in the greatest peak active tension. After L0 was determined, the vessel was set to the optimal length (L0), force was allowed to stabilize in PSS, then the vessel was incubated in a high relaxing (HR) solution for 10–20 min before permeabilization.

Solutions. The HR and pCa solutions used during and following permeabilization of the mesenteric vessels were designed to maintain a desired free Ca2+ concentration, which is presented as pCa values (–log[Ca2+]), using a Ca2+-EGTA buffering system. The composition of the solutions was calculated with the Bathe computer program using the binding constants for all ionic species as reported by Godt and Lindley (14). The HR solution (pCa >9) was composed of the following chemicals (in mM): 53.28 KCl, 6.81 MgCl2, 0.025 CaCl2, 10.0 EGTA, 5.4 Na2ATP, and 12.0 creatine phosphate. The composition of the pCa 4.5 solution was similar to HR, except for the following differences (in mM): 33.74 KCl, 6.48 MgCl2, and 9.96 CaCl2. The pH of the HR and pCa 4.5 solutions were adjusted to 7.0 with KOH, and ionic strength was held constant (0.15). Solutions containing a desired free Ca2+ concentration between pCa 8 and 4.5 were achieved by mixing appropriate volumes of the HR and pCa 4.5 solutions based on the Bathe algorithm. All solutions contained the protease inhibitors leupeptin (1 µg/ml), pepstatin A (2.5 µg/ml), and PMSF (50 µM). The pCa solutions used following β-escin permeabilization of mesenteric lymphatics were supplemented with 1 µM calmodulin (27).

Permeabilization. Mesenteric lymphatics were permeabilized with either {alpha}-toxin from staphylococcus aureus (Calbiochem, San Diego, CA) or β-escin (Sigma, St. Louis, MO). Permeabilization with {alpha}-toxin was performed by incubating the lymphatic segment with 500 U/ml {alpha}-toxin in HR or pCa 6.0 for 30 min at 25°C. The lymphatics permeabilized with β-escin were incubated in 30 µM β-escin in pCa 6.0 for ~5–10 min at 25°C. The mesenteric veins and arteries were permeabilized with {alpha}-toxin (500 U/ml and 1,000 U/ml, respectively) for 20–30 min in HR or pCa 6.25. Preliminary experiments showed no effect of the permeabilization solution (i.e., HR, pCa 6.25 or pCa 6) on subsequent results. Following permeabilization, the vessels were incubated with 10 µM A23187 [GenBank] for 20 min to deplete the intracellular Ca2+ stores (25).

Calcium-tension protocol. After permeabilization, segments were washed several times with HR and then maximally stimulated with pCa 4.5. After this initial activation, the permeabilized preparations were washed several times with HR, and force was allowed to stabilize. The pCa-tension relationship was then determined by bathing the permeabilized vessels in solutions of sequentially increasing Ca2+ concentrations, ranging from pCa 8 to 4.5, while recording force for ~5 min in each solution.

Western blot analysis. Lymphatic vessels were isolated and permeabilized with either {alpha}-toxin or β-escin as described earlier. After permeabilization, the vessels were washed, and the lymphatic samples were sonicated in protein-solubilizing buffer and run on a 4–18% gradient SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane with a Bio-Rad transblot apparatus. The transfer was verified with Ponceau-S staining. Calmodulin or CPI-17 proteins were detected with specific antibodies [Zymed Laboratories, Invitrogen, Carlsbad, CA (1:250 dilution) and Epitomics (Bulingame, CA; 1:1,000 dilution), respectively]. Antibody binding was revealed using the Pierce detection system (SuperSignal West Dura Extended Duration Substrate, Pierce, Rockford, IL). Densitometry on the resulting bands was performed using Multi-Analyst Software (Bio-Rad, Hercules, CA). To verify equal loading of each sample, membranes were stripped using ImmunoPure IgG Elution Buffer (Pierce) and then reprobed with anti-{alpha}-vascular actin primary antibody (1:20,000 dilution; Sigma). The resulting Calmodulin/actin or CPI-17/actin ratio was used for quantitative analyses. Western blot analyses of lymphatic vessel proteins followed by quantification were performed three times for each sample and the resulting mean values ± SE was calculated.

Statistical analysis. Data are presented as mean ± SE. The pCa-tension relationship was normalized to the tension produced in pCa 4.5, then fitted to the Hill equation using nonlinear regression analysis to derive pCa50 (Ca2+ concentration resulting in 50% maximal contraction) and Hill coefficient (nH; slope of the linear portion of the pCa-tension curve that represents cooperative activation) using Prism software. An independent sample t-test was used to determine differences in maximal tension, pCa50 and nH between {alpha}-toxin- and β-escin-permeabilized lymphatics, and between {alpha}-toxin-permeabilized lymphatics and arteries or veins. Significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The results of the length tension and pCa tension protocols for mesenteric lymphatics, arteries and veins are summarized in Table 1. Initially, length-tension relationships were determined prior to the permeabilization procedure in lymphatic vessels. Approximately 80% of the mesenteric lymphatics developed spontaneous phasic contractions at various preloads during assessment of length-tension relations. In those lymphatics that exhibited phasic activity, the force during the relaxation phase of the contraction cycle was used to determine the preload. As described previously (49), mesenteric lymphatics, as well as mesenteric veins, exhibited a biphasic response to maximal activation with KPSS+SP. Figure 1 depicts the length-tension relationship for the peak response of activated mesenteric lymphatics. The maximal peak and plateau active tension was 0.38 ± 0.02 and 0.20 ± 0.02 mN/mm, respectively, and occurred at a preload of 0.09 ± 0.00 mN/mm, which corresponds to a calculated internal pressure of ~17 cmH2O. This is a higher pressure than might be expected based on in vivo or in vitro isobaric preparations, which suggest mesenteric lymphatic pumping peaks at ~5 cmH2O (11), but this phenomenon is fairly consistent with other investigations that have used an isometric preparation to study rat mesenteric lymphatic contractility (48, 49). The slightly higher calculated internal pressure associated with L0 in the current study and that observed previously might be due to the effects of temperature on the mechanical properties in the vessel wall (16), since the present experiments were performed at 25°C, while the earlier studies were performed at ~37°C (11, 48, 49). The plateau phase of the active tension response in mesenteric lymphatics averaged 58 ± 2.5% of the maximal peak active tension and was well maintained over a wide range of preloads (data not shown).


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  		Table 1. Mechanical and contractile characteristics of intact and permeabilized rat mesenteric lymphatics, veins, and arteries

 

Figure 1
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  		Fig. 1. Length-tension relationship of intact mesenteric lymphatics. Tension was calculated as Tension = Force ÷ [2 x vessel length (mm)]; length refers to the circumferential length of the vessel and was calculated based on the distance between the mounting wires and their circumference. ({blacksquare}), wall tension in unstimulated vessels (passive tension); ({square}), wall tension in maximally stimulated vessels (total tension); active tension (—) was calculated as the difference between total and passive tension. Error bars indicate means ± SE; n = 12.

 
After determining L0, mesenteric lymphatics were permeabilized with either {alpha}-toxin or β-escin. Following {alpha}-toxin permeabilization, mesenteric lymphatics exhibited a biphasic time course of force development in response to a given pCa solution (Fig. 2A), which was similar to the response to KPSS+SP demonstrated by intact mesenteric lymphatics (49). In contrast, β-escin-permeabilized mesenteric lymphatics displayed a monophasic force response to an increase in calcium (Fig. 2B). The maximal peak and plateau tension produced by {alpha}-toxin-permeabilized mesenteric lymphatics in pCa 4.5 was 0.47 ± 0.04 and 0.27 ± 0.03 mN/mm, respectively (Fig. 3A). Nonlinear regression analysis of the normalized pCa-tension relationship revealed the pCa50 or the Hill coefficient (nH) for peak and plateau tension values were not significantly different (6.26 ± 0.04 vs. 6.16 ± 0.05, and 2.05 ± 0.23 vs. 1.98 ± 0.19, respectively; Fig. 3B and Table 1). We used the pCa50 and nH values obtained from the pCa-plateau tension relationship of lymphatics to compare with β-escin permeabilized lymphatics or {alpha}-toxin permeabilized artery or veins.


Figure 2
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  		Fig. 2. Representative force tracings of the contractile response of a mesenteric lymphatic after permeabilization with {alpha}-toxin (A) or β-escin (B). A: response of {alpha}-toxin permeabilized lymphatic muscle to submaximal –log[Ca2+] (pCa 6.0) and maximal (pCa 4.5) stimulation; Peak and plateau phases are marked. B: response of β-escin permeabilized lymphatic muscle to submaximal (pCa 5.75) and maximal (pCa 4.5) stimulation.

 

Figure 3
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  		Fig. 3. pCa-tension (A) and normalized pCa-tension (B) relationships of the peak and plateau responses of {alpha}-toxin permeabilized lymphatic muscle. The peak and plateau tension responses for each pCa solution were normalized to the maximal response to pCa 4.5 (B). Data are presented as means ± SE.

 
β-escin permeabilization of lymphatic muscle was associated with significantly lower plateau tension development in pCa 4.5 when compared with {alpha}-toxin-treated lymphatics (0.15 ± 0.01 vs. 0.27 ± 0.03 mN/mm, respectively; P < 0.05; Fig. 4 and Table 1). Therefore, relative to the plateau active tension produced by intact lymphatics at L0, the maximal tension in pCa 4.5 produced by {alpha}-toxin and β-escin permeabilized mesenteric lymphatics was ~135% and ~75%, respectively. The normalized pCa-tension relationship revealed that the pCa50 of {alpha}-toxin permeabilized mesenteric lymphatics was significantly higher compared with that of lymphatics permeabilized with β-escin (6.16 ± 0.05 vs. 5.86 ± 0.06, respectively; P < 0.05; Table 1); however, β-escin permeabilization was associated with a larger nH compared with {alpha}-toxin permeabilized mesenteric lymphatics (3.39 ± 0.39 vs. 1.98 ± 0.19, respectively; P > 0.05; Table 1), which indicates greater myofilament cooperativity in β-escin-permeabilized mesenteric lymphatics.


Figure 4
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  		Fig. 4. pCa-tension relationships of the plateau responses of {alpha}-toxin (n = 10) and β-escin (n = 6) permeabilized lymphatic muscle (A). Normalized pCa-tension relationships of the plateau responses of {alpha}-toxin (n = 10) and β-escin (n = 6) permeabilized lymphatic muscle (B). The tension developed in each pCa solution was normalized to the maximal response in pCa 4.5 (B). Data are presented as means ± SE.

 
Phosphorylation of the MLC20 is the primary mechanism for the regulation of smooth muscle contraction (6, 18). MLC20 phosphorylation is regulated by the relative activity of MLC kinase (MLCK) and MLC phosphatase (MLCP); and the activity of MLCK and MLCP is modulated by the small molecular weight (MW) proteins, calmodulin (CaM; MW = 17) and CPI-17 (MW = 17), respectively (37). Loss of CaM (27) and CPI-17 (26) has been implicated in the depressed contractile response of skinned or stringently permeabilized smooth muscle. To determine whether the decreases in maximal tension and Ca2+ sensitivity following β-escin permeabilization were associated with leakage of CaM and/or CPI-17 from lymphatic muscle, Western blot analysis was performed on intact and {alpha}-toxin- and β-escin-permeabilized mesenteric lymphatics. As shown in Fig. 5, CaM levels were not significantly altered following either permeabilization treatment relative to controls. In contrast, permeabilization of lymphatics with β-escin resulted in an ~50% decrease in CPI-17 levels compared with intact controls (P < 0.05; n = 5–6).


Figure 5
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  		Fig. 5. Representative Western blot results for calmodulin (CaM; A) CPI-17 (B), and smooth muscle (SM) {alpha}-actin (C) from untreated controls (intact) and {alpha}-toxin and β-escin permeabilized lymphatics. SM {alpha}-actin served as an internal control and the ratio of CaM or CPI-17 to SM {alpha}-actin was calculated. CaM:SM {alpha}-actin and CPI-17:SM {alpha}-actin ratios were normalized to the intact treatment (D). *P < 0.05.

 
Since this is the first description of the pCa-tension relationship in permeabilized lymphatic muscle, for comparison, we performed length-tension and pCa-tension experiments in intact and permeabilized mesenteric arteries and veins that lie adjacent to lymphatics. The maximal active tension produced by intact mesenteric arteries was 2.52 ± 0.15 mN/mm and occurred at a preload of 0.76 ± 0.14 mN/mm, which corresponded to a calculated internal pressure of 63.1 ± 8.86 cmH2O. Not surprisingly, the maximal peak active tension produced by mesenteric veins was considerably less than mesenteric arteries (0.63 ± 0.07 mN/mm). The preload at which the maximal response was obtained was 0.25 ± 0.00 mN/mm, which corresponded to a calculated internal pressure of 31.3 ± 1.6 cmH2O. The pCa-tension relationships for {alpha}-toxin-permeabilized mesenteric arterial and venous smooth muscle are presented in Fig. 6. As was observed in lymphatic muscle, both arterial and venous segments generated maximal tensions during pCa 4.5 activation following permeabilization with {alpha}-toxin that were similar to the tensions produced by the intact vessels at L0 (Table 1). The pCa50 of {alpha}-toxin permeabilized mesentery arteries was significantly left-shifted compared with the calcium sensitivity of {alpha}-toxin-treated lymphatic muscle (6.44 ± 0.02 vs. 6.16 ± 0.05; P < 0.05; Fig. 6A and Table 1). In contrast, the pCa50 of {alpha}-toxin permeabilized venous smooth muscle was not significantly different than {alpha}-toxin-permeabilized lymphatic muscle (6.00 ± 0.10 vs. 6.16 ± 0.05; P = 0.12; Fig. 6B and Table 1). These data indicated that the calcium sensitivity of phasic lymphatic muscle is lower than that of tonic arterial smooth muscle, but similar to that of venous smooth muscle. The Hill coefficient (nH), which represents myofilament cooperativity, was significantly higher in lymphatic muscle compared with venous smooth muscle (1.98 ± 0.19 vs. 1.21 ± 0.18; P < 0.05; Fig. 6B and Table 1), but no significant difference was detected between lymphatic and arterial smooth muscle (1.98 ± 0.19 vs. 1.86 ± 0.10; not significant; Fig. 6A and Table 1).


Figure 6
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  		Fig. 6. Normalized pCa-tension relationship of {alpha}-toxin permeabilized mesenteric lymphatic muscle (n = 10) was compared with {alpha}-toxin permeabilized mesenteric arterial (A; n = 6) and venous (B; n = 5) smooth muscle. Tension was normalized to the maximal response elicited by pCa 4.5. Data are presented as means ± SE.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The results presented here are the first demonstration of the pCa-tension relationship in permeabilized lymphatic muscle and, therefore, represent the first detailed investigation of the contractility of the lymphatic myofilament. The data demonstrate that the lymphatic myofilament has contractile characteristics that are unique to this tissue, as indicated by the differences in the calcium sensitivity and cooperative activation compared with arterial and venous myofilaments, respectively, following the same permeabilization treatment. Furthermore, our results demonstrate that the differences in the lymphatic myofilament contractile behavior following two types of permeabilization are related to the loss of a key protein, CPI-17, which modulates contractility.

Our results support the hypothesis that the contractile properties of permeabilized phasic lymphatic muscle differ from that of tonic vascular smooth muscle. The Ca2+ sensitivity of permeabilized phasic lymphatic muscle is approximately twofold lower than that of tonic arterial smooth muscle (Fig. 6 and Table 1). These findings are similar to those of Kitazawa et al. (24), who reported that the Ca2+ sensitivity of {alpha}-toxin-permeabilized phasic vascular (portal vein) and visceral (ileum) smooth muscle is significantly lower that that of tonic arterial smooth muscle. Furthermore, the Ca2+ sensitivity of {alpha}-toxin-permeabilized mesenteric arterial smooth muscle (Table 1) is strikingly similar to that reported for pulmonary and femoral arteries in a previous study (24). Although the pCa50 values of permeabilized lymphatic and venous myofilaments are not significantly different, our data show that the Hill coefficient is significantly greater for the pCa-tension relationship of lymphatic muscle compared with venous smooth muscle. These data indicate that cooperativity among lymphatic myofilament proteins is higher than that in venous smooth muscle. The presented data indicate that the Ca2+-dependent and -independent activation mechanisms of the lymphatic myofilament are distinct from arterial and venous smooth muscle, respectively, which likely underlie important functional differences between these tissues.

Although not measured in the current study, we and others have shown that skinned cardiac muscle generally exhibits a pCa50 close to ~5.8 and a nH close to ~3.0 (9). Thus, the characteristics of {alpha}-toxin permeabilized lymphatic muscle are in between that of striated and smooth muscle myofilaments. Specifically, the pCa50 value of tonic vascular smooth muscle myofilaments is greater than that of lymphatic myofilaments, which is greater than that of cardiac myofilaments (6.44 ± 0.02 > 6.16 ± 0.05 > 5.83 ± 0.02, respectively). However, it should be noted that the pCa50 and nH of β-escin-permeabilized lymphatics were similar to those values normally associated with cardiac muscle (Table 1). These findings are difficult to interpret because a complete characterization of the regulatory proteins associated with the lymphatic myofilament has not been performed. Although we have previously shown that both smooth and striated muscle myosin and actin isoforms are present in lymphatic muscle (33), whether the lymphatic myofilament encompasses regulatory mechanisms ascribed to smooth and striated muscle is an important question, but beyond the scope of the current investigation. However, the results of the present experiments do provide a basis for understanding the molecular mechanisms that regulate lymphatic muscle contraction.

In addition to comparing the pCa-tension relationships between {alpha}-toxin-permeabilized lymphatics and vascular smooth muscle, we also compared the effects of different permeabilization methods on the pCa-tension relationship in lymphatic muscle. Our results indicate that permeabilization of mesenteric lymphatic muscle with {alpha}-toxin and β-escin resulted in striking differences in the contractile response to Ca2+. As shown in Fig. 2, {alpha}-toxin permeabilized mesenteric lymphatics exhibited a biphasic response to submaximal and maximal Ca2+ stimulation, while β-escin permeabilized lymphatics responded to maximal Ca2+ activation with a monophasic contraction. Other laboratories have noted a similar change in the contractile response of phasic ileum smooth muscle to Ca2+ following permeabilization with {alpha}-toxin (biphasic) compared with a monophasic response after permeabilization with β-escin (27) or saponin (25). However, in contrast to the work of Kitazawa et al. (25), the Ca2+ sensitivity associated with the peak response was not greater than the plateau response in {alpha}-toxin-permeabilized lymphatic muscle, thus indicating a subtle difference in the behavior of {alpha}-toxin-permeabilized phasic ileum and lymphatic muscle.

In addition to the altered magnitude and temporal pattern of the contractile response to an increase in Ca2+, the pCa-tension relationship of permeabilized lymphatic muscle was greatly affected by the method of permeabilization. As presented in Table 1 and Fig. 4, {alpha}-toxin-permeabilized mesenteric lymphatics exhibited greater maximal tension and calcium sensitivity than did β-escin-permeabilized lymphatics, whereas myofilament cooperativity was greater following β-escin permeabilization of lymphatic muscle. Our findings differ from those of Watanabe and Takano-Ohmuro (42) and Van Heijs et al. (41), who reported that Ca2+ sensitivity was lower following {alpha}-toxin treatment compared with more stringent permeabilization methods (i.e., β-escin, saponin, or Triton X-100) in guinea pig portal vein and rabbit femoral artery, respectively. Several investigations have reported that proteins smaller than 65 kDa (42), such as calmodulin (27) and CPI-17 (26), may leak out of smooth muscle following β-escin permeabilization, a phenomenon that is often associated with reductions in maximal force and calcium sensitivity (26, 27, 42). Our Western blot data (Fig. 5) demonstrate that β-escin treatment of lymphatics has no significant effect on calmodulin levels but caused CPI-17 levels to be reduced by ~50% relative to untreated controls. It is possible that calmodulin levels were maintained following permeabilization of lymphatics with β-escin because of tight associations between calmodulin and myofilament proteins (43), such as MARCKS (10). Nonetheless, the decrease in CPI-17 levels in lymphatic muscle following β-escin treatment are similar to those of Kitazawa et al. (26) who also reported reductions in CPI-17 levels following β-escin or Triton X-100 treatment of rabbit femoral artery that were also associated with lower force production. In contrast, Ihara et al. (20) found that CPI-17 levels were well maintained in phasic ileum smooth muscle following treatment with β-escin or Triton X-100. These data suggest there are differences in how tightly CPI-17 associates with the myofilaments from different types of smooth muscle. In summary, the lower maximal tension development and calcium sensitivity of lymphatic muscle permeabilized with β-escin is associated with a reduction in CPI-17 protein levels by ~50%.

MLCP is a heterotrimer composed of one catalytic subunit and two regulatory subunits. The catalytic subunit is a type 1 phosphatase {gamma} isoform (PP1c{gamma}) and the larger regulatory subunit, MYPT1, is critical for regulation and localization, while the function of the smaller regulatory subunit remains unclear (for a review, see Ref. 22). In the last 15 years, the regulation of MLCP activity has become increasingly more appreciated in terms of regulating smooth muscle contraction and Ca2+ sensitivity (37). Smooth muscle Ca2+ sensitization primarily occurs through two mechanisms involving the phosphorylation of distinct residues on MYPT1 or CPI-17 (37), which leads to the inhibition of MLCP. CPI-17 is a potent and selective inhibitor of MLCP and its inhibitory influence is greatly increased upon phosphorylation of its Thr38 residue (34). CPI-17 is considered a point of convergence since numerous signaling pathways, including PKC (7, 23), ROK (7, 23), and ILK (5), have been shown to induce smooth muscle contraction and Ca2+ sensitization via CPI-17-dependent inhibition of MLCP. Interestingly, Kitazawa et al. (26) reported that changes in Ca2+ sensitivity were associated with reductions in CPI-17 levels in β-escin and Triton X-100 skinned smooth muscle. Furthermore, reconstitution of CPI-17 and its potent activator, PKC{alpha} enhanced the Ca2+ sensitivity in Triton X-100-skinned smooth muscle (26). Woodsome et al. (44) reported that expression of CPI-17 correlates with Ca2+ sensitization via PKC in tonic and phasic smooth muscle. Thus, our data showing a decrease in Ca2+ sensitivity following β-escin permeabilization of lymphatic muscle in association with a loss of CPI-17 protein are consistent with several reports in the literature. Future studies are warranted to determine the signaling pathways that modulate lymphatic muscle contractility and Ca2+ sensitivity through CPI-17- and MYPT1-dependent regulation of MLCP.

Perspectives and Significance

Our data demonstrate that the pCa-tension relationship of permeabilized phasic lymphatic muscle is different from those of tonic vascular smooth muscle. We postulate that the fundamental differences in myofilament activation between these tissues contribute to their specialized functions in regulating blood pressure (arteries) and volume (veins) and in the generation and regulation of lymph flow (lymphatics). In addition, we provide indirect evidence that CPI-17-dependent inhibition of MLCP is an important regulatory mechanism of lymphatic muscle contraction and Ca2+ sensitivity. The use of these permeabilized lymphatic muscle preparations will accelerate our understanding of the unique contractile properties of this tissue. Furthermore, these types of experiments will allow us to determine whether alterations in the lymphatic myofilament contribute to impaired function of the lymph pump in disease conditions such as chronic edema.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by grants from National Institutes of Health HL-080526 and KO2 HL086650 to M. Muthuchamy, and HL-75199 to D. Zawieja.


   ACKNOWLEDGMENTS
 
The authors would like to thank Scott Zawieja for his help in preparing calcium solutions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Muthuchamy, Dept. of Systems Biology and Translational Medicine, Texas A&M Health Science Center College of Medicine, College Station, TX (e-mail: marim{at}tamu.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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