calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics

doi:10.1152/ajpregu.00888.2007 294:1524-1532, 2008. First published Feb 27, 2008;Am J Physiol Regulatory Integrative Comp Physiol

Muthuchamy Patrick J. Dougherty, Michael J. Davis, David C. Zawieja and Mariappan

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Calcium sensitivity and cooperativity of permeabilized ratmesenteric 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 LymphaticBiology, Texas A&M Health Science Center College of Medicine, College Station, Texas; and 2Department of MedicalPharmacology and Physiology, University of Missouri School of Medicine, Columbia, Missouri

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

Dougherty PJ, Davis MJ, Zawieja DC, Muthuchamy M. Calciumsensitivity and cooperativity of permeabilized rat mesenteric lymphatics.Am J Physiol Regul Integr Comp Physiol 294: R1524–R1532, 2008. Firstpublished February 27, 2008; doi:10.1152/ajpregu.00888.2007.—Lym-phatic muscle contraction is critical for the centripetal movement oflymph that regulates fluid balance, protein homeostasis, lipid absorp-tion, and immune function. We have demonstrated that lymphaticmuscle has both smooth and striated muscle contractile elements;however, the basic contractile properties of this tissue remain poorlydefined. We hypothesized that contractile characteristics of lymphaticmyofilaments would be different from vascular smooth muscle myo-filaments. To test this hypothesis, �log[Ca2�] (pCa)-tension relation-ship was determined for �-toxin permeabilized mesenteric lymphat-ics, arteries, and veins. The Ca2� sensitivity (pCa50) of mesentericlymphatics was significantly lower compared with arteries (6.16 �0.05 vs. 6.44 � 0.02; P � 0.05), whereas there was no difference inpCa50 between lymphatics and veins (6.16 � 0.05 vs. 6.00 � 0.10; notsignificant). The Hill coefficient for �-toxin-permeabilized lymphaticswas not significantly different from arteries but was significantlygreater 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 significantlygreater in �-toxin-permeabilized lymphatics compared with �-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 theHill coefficient was significantly greater in �-escin-permeabilizedlymphatics. Western blot analyses revealed that CPI-17 levels weresignificantly decreased by about 50% in �-escin-permeabilized lym-phatics, compared with controls, whereas no change in the level ofcalmodulin was detected. Our data constitute the first description ofthe pCa-tension relationship in permeabilized lymphatic muscle. Itsuggests that differences in myofilament Ca2� sensitivity and coop-erativity among lymphatic muscle and vascular smooth musclescontribute to the functional differences that exist between thesetissues.

lymphatic muscle; pCa-force relationship; calcium sensitivity

THE LYMPHATIC VASCULAR SYSTEM plays an important role in fluidand protein homeostasis, lipid absorption, and immune func-tion (47). Lymphatic transport of fluids, macromolecules, andimmune cells from the peripheral tissues to the blood vascularsystem occurs against a net pressure gradient. Thus, lymphtransport relies on a system of lymph pumps and valves todrive and direct lymph flow. Impairment of the lymph pumphas been documented in several pathological conditions, suchas aging (13), inflammatory bowel disease (45), and in womenfollowing lymph node resection to treat breast cancer (32), and

is associated with lymphedema and an increased risk of infec-tion in the affected region (28, 36). Despite its critical role inhealth and disease, relatively little is known about basic con-tractile properties of lymphatic muscle (47).

Extrinsic and intrinsic mechanisms contribute to the move-ment of lymph through the lymphatic system. Initial lymphat-ics do not possess muscle cells and rely upon extrinsic factors,such as mechanical compression from skeletal muscle, to movelymph downstream into collecting lymphatics, which are linedwith one or more layers of muscle cells. Collecting lymphaticspossess a patent one-way valve system that promotes unidirec-tional flow and divides the collecting vessels into smallerfunctional units, termed lymphangions. The intrinsic lymphpump refers to the strong, brisk phasic contractions of lym-phatic muscle that serve to propel lymph into downstreamlymphangions, while the patent one-way valves preventretrograde lymph flow. The phasic contractions of lymphaticmuscle are initiated by a depolarizing action potential andare associated with transient calcium spikes. Because col-lecting lymphatics exhibit distinct cycles of systole anddiastole, the lymph pump is often analyzed using cardiacfunction indices (3).

Lymphatic muscle is a highly specialized type of smoothmuscle that exhibits both tonic and phasic contractions that areresponsible for the distinct periods of diastole and systole,respectively, observed in collecting lymphatics. Both tonic andphasic contractions are sensitive to changes in pressure andflow (11, 12), extracellular Ca2� concentration (29), and ago-nist stimulation (1, 2). The phasic contractions in rat mesen-teric lymphatics occur at frequencies of �5–15 contractionsper minute (48). Interestingly, the shortening velocity of lym-phatic muscle has been reported to be �2- and �5-fold fasterthan that of phasic and tonic smooth muscle, respectively, andcloser to that of striated muscle (3). In support of the uniquefunctional behavior of lymphatic muscle, we have recentlyreported that the lymphatic muscle contractile protein (i.e.,actin and myosin) expression profile is composed of bothcardiac/skeletal and smooth muscle isoforms (33). Our resultsdemonstrated that rat mesenteric lymphatics express only smoothmuscle B (SMB) smooth muscle myosin heavy chain (SM-MHC), whereas, nearby mesenteric arterioles expressed bothsmooth muscle A (SMA) and SMB isoforms. In addition,slow-skeletal/fetal cardiac muscle-specific �-MHC messagewas detected only in mesenteric lymphatics. Furthermore, allfour actin messages, �-cardiac, �-vascular, �-enteric, and �-skel-

Address for reprint requests and other correspondence: M. Muthuchamy,Dept. of Systems Biology and Translational Medicine, Texas A&M HealthScience Center College of Medicine, College Station, TX (e-mail: [email protected]).

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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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etal, were present in both mesenteric lymphatics and arterioles.Western blot and immunohistochemical analyses corroboratedthe mRNA studies with the exception that only �-vascularactin protein was detected in arterioles. This combination ofsmooth and striated muscle contractile elements present inlymphatic muscle could provide unique contractile character-istics (i.e., Ca2� sensitivity and cooperativity) for lymphaticmyofilaments. Although numerous mechanical (3, 12, 30, 35,49) and neurohumoral (1, 2, 19, 31, 46, 50) factors have beenshown to modulate the tonic and phasic activity of collectinglymphatics, few investigations of the basic contractile proper-ties of the lymphatic myofilament have been published.

Smooth muscle is generally classified as either tonic orphasic based on its contractile behavior (38). Regulation ofsmooth muscle contraction occurs primarily through the phos-phorylation of the 20-kDa regulatory myosin light chain(MLC20), which is sensitive to the relative activities of MLCkinase (MLCK) and MLC phosphatase (MLCP) (37). A num-ber of published reports have indicated that significant differ-ences exist between the molecular composition and the con-tractility of myofilaments from tonic and phasic smooth mus-cle. For example, phasic smooth muscle expresses higherlevels of the acidic 17-kDa myosin light chain isoform (39), theMLCP regulatory subunit, MYPT1, (44), and the thin-filamentproteins caldesmon and calmodulin (40). Reports indicate thatboth MLCK and MLCP activities are higher in phasic com-pared 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 inhib-itor protein of 17 kDa), which is a potent inhibitor of MLCPwhen activated via phosphorylation of Thr38 (17). In additionto the molecular differences between tonic and phasic smoothmuscle myofilaments, tonic vascular smooth muscle treatedwith the mild permeabilizing agent, �-toxin, has been shown toexhibit significantly greater Ca2� sensitivity than does phasicvascular and visceral smooth muscle (24). Although detailedcomparisons have been made between the myofilaments fromtonic and phasic smooth muscle from different organ systems,no such data are available for lymphatic muscle, which exhibitsboth tonic and phasic activities.

In recent studies characterizing the mechanical properties oflymphatic muscle, we have shown that mesenteric lymphaticmuscle exhibits striking contractile differences when comparedwith small arteries and veins from the same vascular bed.Though lymphatic vessels generate the least active tension andstress among these three vessels, the lymphatic vessels exhib-ited distinct elastic characteristics, as well as both phasic andtonic contractions (4, 48, 49). Because tonic and phasic smoothmuscle myofilaments exhibit different biochemical character-istics and the lymphatic muscle has unique biomechanicalproperties, we hypothesized that the calcium sensitivity andcooperativity of lymphatic myofilaments must be differentfrom tonic vascular smooth muscle to attain its unique phasicand tonic contractile nature. To test this hypothesis, we deter-mined the calcium-tension relationship in permeabilized mes-enteric lymphatic segments and compared it with the calcium-tension relationships of permeabilized mesenteric arteries andveins.

The use of permeabilized smooth muscle preparations pro-vides important information regarding basic biochemical prop-erties, such as calcium sensitivity (pCa50) and cooperativity

(nH) of contraction at the myofilament level. There are severalmethods frequently used to permeabilize or skin smooth mus-cle; these include �-toxin, �-escin, saponin, and Triton X-100.Treatment with �-toxin or �-escin results in permeabilizedmembranes that maintain receptor-coupled signaling pathways(25, 27) that are disrupted after treatment with saponin orTriton X-100. For this reason, �-toxin- or �-escin-permeabil-ized preparations have been critical in determining the complexinteractions between agonist stimulation and myofilament ac-tivation in smooth muscle. However, the size of the porescreated by �-toxin and �-escin differs substantially. Permeabi-lization with �-toxin results in the formation of small trans-membrane pores that allow the passage of ions and smallmolecules but limits the movement of large molecules (8),whereas �-escin creates larger pores that allow passage of ionsand small and larger molecules across the permeabilized mem-brane (21). Because of the advantages and disadvantagesinherent in the different permeabilization methods and theconflicting reports regarding the effects of permeabilizationtreatment on smooth muscle contractility (41, 42), we alsodetermined the effects of two types of permeabilization (�-toxin and �-escin) on the calcium-tension relationship of per-meabilized lymphatic muscle.

MATERIALS AND METHODS

Animals and procedures. Male Sprague-Dawley rats (250 – 400 g)were anesthetized with intramuscular injections of diazepam (2.5mg/kg body wt) and a fentanyl/droperidol solution (0.3 ml/kg). Amidline incision was made through the skin, fascia, and underlyingabdominal muscle layers, and a 6- to 7-cm loop of the smallintestine was exteriorized. With the assistance of a stereomicro-scope (Olympus SZX12, Leeds Instruments, Irving, TX), a suitablemesenteric artery, vein, or lymphatic was identified and carefullycleaned of fat and connective tissue, while being continuouslysuperfused with PBS. A 1-cm-long segment of the vessel to bestudied was dissected free and transferred to a 35-mm Petri dishcontaining PBS. After the tissue was removed, the animal waskilled by cervical dislocation. All animal procedures were re-viewed and approved by the Texas A&M University LaboratoryAnimal Care Committee and adhered to both institutional andfederal guidelines.

Isolated vessel procedures. Two stainless steel wires (40 m ODfor arteries and veins; 25 m OD for lymphatics) were passed, one ata time, through the lumen of a 2-mm segment of the isolatedmesenteric vessel. The vessel was then transferred to the organchamber of a single-channel, wire myograph (Model 310A, DanishMyo Technology, Aarhus, Denmark) that contained a physiologicalsalt solution (PSS) of the following composition (in mM): 145.0NaCl, 4.7 KCl, 2 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0sodium pyruvate, 0.02 EDTA and 3.0 3-(N-morpholino) propanesul-fonic acid (MOPS). One wire was secured to the jaw of the myographthat was under control of an adjustable micrometer, which was usedto stretch the vessel between the two parallel wires. The other wirewas secured to the other jaw of the myograph coupled to a calibratedforce transducer (Danish Myo Technology). The force transducermeasured forces between 0 and 30 mN and the force output wasdigitized with a PCI-6030e A–D card and interface (National Instru-ments, Austin, TX). Experiments were recorded using LabView(National Instruments) at a sampling frequency of 10 Hz and stored ona 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 CCDcamera (Model PK-M2U, Hitachi Denshi, Woodbury, NY). A videomicrometer (Microcirculation Research Institute, Texas A&M Uni-

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versity, College Station, TX) was used to manually measure innerdiameter.

Length-tension protocol. A vessel segment was mounted in themyograph, and the bathing solution was maintained at 25°C. Theexperimental protocols were initiated after a 30- to 60-min equilibra-tion period during which the bathing solution was changed once. Wefirst attested the length-activated tension relationship of each vessel,as described previously (49). Briefly, vessels were stretched to a rangeof predetermined preloads. After a steady preload was achieved,vessels were maximally activated with KPSS (PSS with equimolarsubstitution of KCl for NaCl). For the experiments with mesentericlymphatics and veins, the KPSS was supplemented with 1 MSubstance P (SP; KPSS�SP). The optimal circumferential length (L0)was defined as the internal circumference that resulted in the greatestpeak active tension. After L0 was determined, the vessel was set to theoptimal length (L0), force was allowed to stabilize in PSS, then thevessel was incubated in a high relaxing (HR) solution for 10–20 minbefore permeabilization.

Solutions. The HR and pCa solutions used during and followingpermeabilization of the mesenteric vessels were designed to maintaina desired free Ca2� concentration, which is presented as pCa values(�log[Ca2�]), using a Ca2�-EGTA buffering system. The composi-tion of the solutions was calculated with the Bathe computer programusing the binding constants for all ionic species as reported by Godtand Lindley (14). The HR solution (pCa 9) was composed of thefollowing chemicals (in mM): 53.28 KCl, 6.81 MgCl2, 0.025 CaCl2,10.0 EGTA, 5.4 Na2ATP, and 12.0 creatine phosphate. The compo-sition of the pCa 4.5 solution was similar to HR, except for thefollowing differences (in mM): 33.74 KCl, 6.48 MgCl2, and 9.96CaCl2. The pH of the HR and pCa 4.5 solutions were adjusted to 7.0with KOH, and ionic strength was held constant (0.15). Solutionscontaining a desired free Ca2� concentration between pCa 8 and 4.5were achieved by mixing appropriate volumes of the HR and pCa 4.5solutions based on the Bathe algorithm. All solutions contained theprotease inhibitors leupeptin (1 g/ml), pepstatin A (2.5 g/ml), andPMSF (50 M). The pCa solutions used following �-escin permeabi-lization of mesenteric lymphatics were supplemented with 1 Mcalmodulin (27).

Permeabilization. Mesenteric lymphatics were permeabilized witheither �-toxin from staphylococcus aureus (Calbiochem, San Diego,CA) or �-escin (Sigma, St. Louis, MO). Permeabilization with�-toxin was performed by incubating the lymphatic segment with 500U/ml �-toxin in HR or pCa 6.0 for 30 min at 25°C. The lymphaticspermeabilized with �-escin were incubated in 30 M �-escin in pCa6.0 for �5–10 min at 25°C. The mesenteric veins and arteries werepermeabilized with �-toxin (500 U/ml and 1,000 U/ml, respectively)for 20–30 min in HR or pCa 6.25. Preliminary experiments showedno effect of the permeabilization solution (i.e., HR, pCa 6.25 or pCa6) on subsequent results. Following permeabilization, the vessels wereincubated with 10 M A23187 for 20 min to deplete the intracellularCa2� stores (25).

Calcium-tension protocol. After permeabilization, segments werewashed several times with HR and then maximally stimulated withpCa 4.5. After this initial activation, the permeabilized preparationswere washed several times with HR, and force was allowed tostabilize. The pCa-tension relationship was then determined by bath-ing the permeabilized vessels in solutions of sequentially increasingCa2� concentrations, ranging from pCa 8 to 4.5, while recording forcefor �5 min in each solution.

Western blot analysis. Lymphatic vessels were isolated and per-meabilized with either �-toxin or �-escin as described earlier. Afterpermeabilization, the vessels were washed, and the lymphatic sampleswere sonicated in protein-solubilizing buffer and run on a 4–18%gradient SDS-polyacrylamide gel. The proteins were transferred to anitrocellulose membrane with a Bio-Rad transblot apparatus. Thetransfer was verified with Ponceau-S staining. Calmodulin or CPI-17proteins 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 revealedusing the Pierce detection system (SuperSignal West Dura ExtendedDuration Substrate, Pierce, Rockford, IL). Densitometry on the result-ing bands was performed using Multi-Analyst Software (Bio-Rad,Hercules, CA). To verify equal loading of each sample, membraneswere stripped using ImmunoPure IgG Elution Buffer (Pierce) and thenreprobed with anti-�-vascular actin primary antibody (1:20,000 dilu-tion; Sigma). The resulting Calmodulin/actin or CPI-17/actin ratiowas used for quantitative analyses. Western blot analyses of lymphaticvessel proteins followed by quantification were performed three timesfor 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 pCa4.5, then fitted to the Hill equation using nonlinear regression analysisto derive pCa50 (Ca2� concentration resulting in 50% maximal con-traction) and Hill coefficient (nH; slope of the linear portion of thepCa-tension curve that represents cooperative activation) using Prismsoftware. An independent sample t-test was used to determine differ-ences in maximal tension, pCa50 and nH between �-toxin- and�-escin-permeabilized lymphatics, and between �-toxin-permeabil-ized lymphatics and arteries or veins. Significance was set atP � 0.05.

RESULTS

The results of the length tension and pCa tension protocolsfor mesenteric lymphatics, arteries and veins are summarizedin Table 1. Initially, length-tension relationships were deter-mined prior to the permeabilization procedure in lymphaticvessels. Approximately 80% of the mesenteric lymphaticsdeveloped spontaneous phasic contractions at various preloadsduring assessment of length-tension relations. In those lym-

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

Lymphatic Artery Vein

IntactDiameter at L0, m 105.9�0.00 255.0�0.01 183.1�0.01Passive tension at L0, mN/mm 0.09�0.00 0.76�0.14 0.25�0.00Pressure at L0, cmH2O 17.5�0.66 63.1�8.86 31.3�1.6Maximal peak active tension,

mN/mm 0.38�0.02 2.52�0.15 0.63�0.07Maximal plateau active

tension, mN/mm 0.20�0.02 0.15�0.04

Permeabilization

Lymphatic Artery Vein

�-Toxin �-Escin �-Toxin �-Toxin

Maximal peaktension, mN/mm 0.47�0.04*

Maximal plateautension, mN/mm 0.27�0.03 0.15�0.01* 2.55�0.16* 0.59�0.10*

pCa50 6.16�0.05 5.86�0.06* 6.44�0.02* 6.00�0.10nH 1.98�0.19 3.39�0.39* 1.86�0.10 1.21�0.18*

Data are presented as means � SE. Diameter was calculated from theinternal circumference of the vessel at L0 assuming a cylindrical geometry.Tension was calculated by dividing force by two times the vessel length. ThepCa-tension relationship was normalized to the tension developed in responseto pCa 4.5. The pCa50 and Hill coefficient values were obtained by fitting thepCa-tension relationship to the Hill equation. The pCa50 represents the pCavalue (Ca2� concentration) at which 50% of the maximal contraction occurred.The Hill coefficient (nH) represents the slope of the linear portion of thepCa-tension relationship. *Significant difference from the corresponding pla-teau response value of �-toxin-permeabilized mesenteric lymphatics.

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phatics that exhibited phasic activity, the force during therelaxation phase of the contraction cycle was used to determinethe preload. As described previously (49), mesenteric lymphatics,as well as mesenteric veins, exhibited a biphasic response tomaximal activation with KPSS�SP. Figure 1 depicts thelength-tension relationship for the peak response of activatedmesenteric lymphatics. The maximal peak and plateau activetension was 0.38 � 0.02 and 0.20 � 0.02 mN/mm, respec-tively, and occurred at a preload of 0.09 � 0.00 mN/mm,which corresponds to a calculated internal pressure of �17cmH2O. This is a higher pressure than might be expected basedon in vivo or in vitro isobaric preparations, which suggestmesenteric lymphatic pumping peaks at �5 cmH2O (11), butthis phenomenon is fairly consistent with other investigationsthat have used an isometric preparation to study rat mesentericlymphatic contractility (48, 49). The slightly higher calculatedinternal pressure associated with L0 in the current study andthat observed previously might be due to the effects of tem-

perature on the mechanical properties in the vessel wall (16),since the present experiments were performed at 25°C, whilethe earlier studies were performed at �37°C (11, 48, 49). Theplateau phase of the active tension response in mesentericlymphatics averaged 58 � 2.5% of the maximal peak activetension and was well maintained over a wide range of preloads(data not shown).

After determining L0, mesenteric lymphatics were perme-abilized with either �-toxin or �-escin. Following �-toxinpermeabilization, mesenteric lymphatics exhibited a biphasictime course of force development in response to a given pCasolution (Fig. 2A), which was similar to the response toKPSS�SP demonstrated by intact mesenteric lymphatics (49).In contrast, �-escin-permeabilized mesenteric lymphatics dis-played a monophasic force response to an increase in calcium(Fig. 2B). The maximal peak and plateau tension produced by�-toxin-permeabilized mesenteric lymphatics in pCa 4.5 was0.47 � 0.04 and 0.27 � 0.03 mN/mm, respectively (Fig. 3A).Nonlinear regression analysis of the normalized pCa-tensionrelationship revealed the pCa50 or the Hill coefficient (nH) forpeak 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 oflymphatics to compare with �-escin permeabilized lymphaticsor �-toxin permeabilized artery or veins.

�-escin permeabilization of lymphatic muscle was associ-ated with significantly lower plateau tension development inpCa 4.5 when compared with �-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 activetension produced by intact lymphatics at L0, the maximaltension in pCa 4.5 produced by �-toxin and �-escin perme-abilized mesenteric lymphatics was �135% and �75%, re-spectively. The normalized pCa-tension relationship revealedthat the pCa50 of �-toxin permeabilized mesenteric lymphaticswas significantly higher compared with that of lymphaticspermeabilized with �-escin (6.16 � 0.05 vs. 5.86 � 0.06,respectively; P � 0.05; Table 1); however, �-escin permeabi-lization was associated with a larger nH compared with �-toxin

Fig. 1. Length-tension relationship of intact mesenteric lymphatics. Tensionwas calculated as Tension � Force � [2 vessel length (mm)]; length refersto the circumferential length of the vessel and was calculated based on thedistance between the mounting wires and their circumference. (■ ), wall tensionin unstimulated vessels (passive tension); (�), wall tension in maximallystimulated vessels (total tension); active tension (—) was calculated as thedifference between total and passive tension. Error bars indicate means �SE; n � 12.

Fig. 2. Representative force tracings of the contractileresponse of a mesenteric lymphatic after permeabiliza-tion with �-toxin (A) or �-escin (B). A: response of�-toxin permeabilized lymphatic muscle to submaximal�log[Ca2�] (pCa 6.0) and maximal (pCa 4.5) stimula-tion; Peak and plateau phases are marked. B: responseof �-escin permeabilized lymphatic muscle to submaxi-mal (pCa 5.75) and maximal (pCa 4.5) stimulation.



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permeabilized mesenteric lymphatics (3.39 � 0.39 vs. 1.98 �0.19, respectively; P 0.05; Table 1), which indicates greatermyofilament cooperativity in �-escin-permeabilized mesen-teric lymphatics.

Phosphorylation of the MLC20 is the primary mechanism forthe regulation of smooth muscle contraction (6, 18). MLC20

phosphorylation is regulated by the relative activity of MLCkinase (MLCK) and MLC phosphatase (MLCP); and the ac-tivity of MLCK and MLCP is modulated by the small molec-ular weight (MW) proteins, calmodulin (CaM; MW � 17) andCPI-17 (MW � 17), respectively (37). Loss of CaM (27) andCPI-17 (26) has been implicated in the depressed contractileresponse of skinned or stringently permeabilized smooth mus-cle. To determine whether the decreases in maximal tensionand Ca2� sensitivity following �-escin permeabilization wereassociated with leakage of CaM and/or CPI-17 from lymphaticmuscle, Western blot analysis was performed on intact and�-toxin- and �-escin-permeabilized mesenteric lymphatics. Asshown in Fig. 5, CaM levels were not significantly alteredfollowing either permeabilization treatment relative to con-trols. In contrast, permeabilization of lymphatics with �-escinresulted in an �50% decrease in CPI-17 levels compared withintact controls (P � 0.05; n � 5–6).

Since this is the first description of the pCa-tension relation-ship in permeabilized lymphatic muscle, for comparison, weperformed length-tension and pCa-tension experiments in in-tact and permeabilized mesenteric arteries and veins that lieadjacent to lymphatics. The maximal active tension producedby intact mesenteric arteries was 2.52 � 0.15 mN/mm andoccurred at a preload of 0.76 � 0.14 mN/mm, which corre-sponded to a calculated internal pressure of 63.1 � 8.86cmH2O. Not surprisingly, the maximal peak active tensionproduced by mesenteric veins was considerably less thanmesenteric arteries (0.63 � 0.07 mN/mm). The preload atwhich the maximal response was obtained was 0.25 � 0.00mN/mm, which corresponded to a calculated internal pressure

of 31.3 � 1.6 cmH2O. The pCa-tension relationships for�-toxin-permeabilized mesenteric arterial and venous smoothmuscle are presented in Fig. 6. As was observed in lymphaticmuscle, both arterial and venous segments generated maximaltensions during pCa 4.5 activation following permeabilizationwith �-toxin that were similar to the tensions produced by theintact vessels at L0 (Table 1). The pCa50 of �-toxin permeabil-ized mesentery arteries was significantly left-shifted comparedwith the calcium sensitivity of �-toxin-treated lymphatic mus-cle (6.44 � 0.02 vs. 6.16 � 0.05; P � 0.05; Fig. 6A and Table1). In contrast, the pCa50 of �-toxin permeabilized venoussmooth muscle was not significantly different than �-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 thecalcium sensitivity of phasic lymphatic muscle is lower thanthat of tonic arterial smooth muscle, but similar to that ofvenous smooth muscle. The Hill coefficient (nH), which rep-resents myofilament cooperativity, was significantly higher inlymphatic 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 lymphaticand arterial smooth muscle (1.98 � 0.19 vs. 1.86 � 0.10; notsignificant; Fig. 6A and Table 1).

DISCUSSION

The results presented here are the first demonstration of thepCa-tension relationship in permeabilized lymphatic muscleand, therefore, represent the first detailed investigation of thecontractility of the lymphatic myofilament. The data demon-strate that the lymphatic myofilament has contractile charac-teristics that are unique to this tissue, as indicated by thedifferences in the calcium sensitivity and cooperative activa-tion compared with arterial and venous myofilaments, respec-tively, following the same permeabilization treatment. Further-more, our results demonstrate that the differences in the lym-

Fig. 3. pCa-tension (A) and normalized pCa-tension (B) relationships of the peak and plateauresponses of �-toxin permeabilized lymphaticmuscle. The peak and plateau tension responsesfor each pCa solution were normalized to themaximal response to pCa 4.5 (B). Data arepresented as means � SE.

Fig. 4. pCa-tension relationships of the plateauresponses of �-toxin (n � 10) and �-escin (n �6) permeabilized lymphatic muscle (A). Normal-ized pCa-tension relationships of the plateauresponses of �-toxin (n � 10) and �-escin (n �6) permeabilized lymphatic muscle (B). Thetension developed in each pCa solution wasnormalized to the maximal response in pCa 4.5(B). Data are presented as means � SE.



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phatic myofilament contractile behavior following two types ofpermeabilization are related to the loss of a key protein,CPI-17, which modulates contractility.

Our results support the hypothesis that the contractile prop-erties of permeabilized phasic lymphatic muscle differ fromthat of tonic vascular smooth muscle. The Ca2� sensitivity ofpermeabilized phasic lymphatic muscle is approximately two-fold lower than that of tonic arterial smooth muscle (Fig. 6 andTable 1). These findings are similar to those of Kitazawa et al.(24), who reported that the Ca2� sensitivity of �-toxin-perme-abilized phasic vascular (portal vein) and visceral (ileum)smooth muscle is significantly lower that that of tonic arterialsmooth muscle. Furthermore, the Ca2� sensitivity of �-toxin-permeabilized mesenteric arterial smooth muscle (Table 1) isstrikingly similar to that reported for pulmonary and femoralarteries in a previous study (24). Although the pCa50 values ofpermeabilized lymphatic and venous myofilaments are notsignificantly different, our data show that the Hill coefficient issignificantly greater for the pCa-tension relationship of lym-phatic muscle compared with venous smooth muscle. Thesedata indicate that cooperativity among lymphatic myofilamentproteins is higher than that in venous smooth muscle. Thepresented data indicate that the Ca2�-dependent and -indepen-dent activation mechanisms of the lymphatic myofilament aredistinct from arterial and venous smooth muscle, respectively,

which likely underlie important functional differences betweenthese tissues.

Although not measured in the current study, we and othershave shown that skinned cardiac muscle generally exhibits apCa50 close to �5.8 and a nH close to �3.0 (9). Thus, thecharacteristics of �-toxin permeabilized lymphatic muscle arein between that of striated and smooth muscle myofilaments.Specifically, the pCa50 value of tonic vascular smooth musclemyofilaments 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, itshould be noted that the pCa50 and nH of �-escin-permeabil-ized lymphatics were similar to those values normally associ-ated with cardiac muscle (Table 1). These findings are difficultto interpret because a complete characterization of the regula-tory proteins associated with the lymphatic myofilament hasnot been performed. Although we have previously shown thatboth smooth and striated muscle myosin and actin isoforms arepresent in lymphatic muscle (33), whether the lymphatic myo-filament encompasses regulatory mechanisms ascribed tosmooth and striated muscle is an important question, butbeyond the scope of the current investigation. However, theresults of the present experiments do provide a basis forunderstanding the molecular mechanisms that regulate lym-phatic muscle contraction.

Fig. 5. Representative Western blot results forcalmodulin (CaM; A) CPI-17 (B), and smoothmuscle (SM) �-actin (C) from untreated controls(intact) and �-toxin and �-escin permeabilizedlymphatics. SM �-actin served as an internalcontrol and the ratio of CaM or CPI-17 to SM�-actin was calculated. CaM:SM �-actin andCPI-17:SM �-actin ratios were normalized tothe intact treatment (D). *P � 0.05.

Fig. 6. Normalized pCa-tension relationship of�-toxin permeabilized mesenteric lymphaticmuscle (n � 10) was compared with �-toxinpermeabilized mesenteric arterial (A; n � 6) andvenous (B; n � 5) smooth muscle. Tension wasnormalized to the maximal response elicited bypCa 4.5. Data are presented as means � SE.



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In addition to comparing the pCa-tension relationships be-tween �-toxin-permeabilized lymphatics and vascular smoothmuscle, we also compared the effects of different permeabili-zation methods on the pCa-tension relationship in lymphaticmuscle. Our results indicate that permeabilization of mesen-teric lymphatic muscle with �-toxin and �-escin resulted instriking differences in the contractile response to Ca2�. Asshown in Fig. 2, �-toxin permeabilized mesenteric lymphaticsexhibited a biphasic response to submaximal and maximalCa2� stimulation, while �-escin permeabilized lymphatics re-sponded to maximal Ca2� activation with a monophasic con-traction. Other laboratories have noted a similar change in thecontractile response of phasic ileum smooth muscle to Ca2�

following permeabilization with �-toxin (biphasic) comparedwith a monophasic response after permeabilization with �-es-cin (27) or saponin (25). However, in contrast to the work ofKitazawa et al. (25), the Ca2� sensitivity associated with thepeak response was not greater than the plateau response in�-toxin-permeabilized lymphatic muscle, thus indicating asubtle difference in the behavior of �-toxin-permeabilizedphasic ileum and lymphatic muscle.

In addition to the altered magnitude and temporal pattern ofthe contractile response to an increase in Ca2�, the pCa-tensionrelationship of permeabilized lymphatic muscle was greatlyaffected by the method of permeabilization. As presented inTable 1 and Fig. 4, �-toxin-permeabilized mesenteric lymphat-ics exhibited greater maximal tension and calcium sensitivitythan did �-escin-permeabilized lymphatics, whereas myofila-ment cooperativity was greater following �-escin permeabili-zation of lymphatic muscle. Our findings differ from those ofWatanabe and Takano-Ohmuro (42) and Van Heijs et al. (41),who reported that Ca2� sensitivity was lower following�-toxin treatment compared with more stringent permeabiliza-tion methods (i.e., �-escin, saponin, or Triton X-100) in guineapig portal vein and rabbit femoral artery, respectively. Severalinvestigations have reported that proteins smaller than 65 kDa(42), such as calmodulin (27) and CPI-17 (26), may leak out ofsmooth muscle following �-escin permeabilization, a phenom-enon that is often associated with reductions in maximal forceand calcium sensitivity (26, 27, 42). Our Western blot data(Fig. 5) demonstrate that �-escin treatment of lymphatics hasno significant effect on calmodulin levels but caused CPI-17levels to be reduced by �50% relative to untreated controls. Itis possible that calmodulin levels were maintained followingpermeabilization of lymphatics with �-escin because of tightassociations between calmodulin and myofilament proteins(43), such as MARCKS (10). Nonetheless, the decrease inCPI-17 levels in lymphatic muscle following �-escin treatmentare similar to those of Kitazawa et al. (26) who also reportedreductions in CPI-17 levels following �-escin or Triton X-100treatment of rabbit femoral artery that were also associatedwith lower force production. In contrast, Ihara et al. (20) foundthat CPI-17 levels were well maintained in phasic ileumsmooth muscle following treatment with �-escin or TritonX-100. These data suggest there are differences in how tightlyCPI-17 associates with the myofilaments from different typesof smooth muscle. In summary, the lower maximal tensiondevelopment and calcium sensitivity of lymphatic muscle per-meabilized with �-escin is associated with a reduction inCPI-17 protein levels by �50%.

MLCP is a heterotrimer composed of one catalytic subunitand two regulatory subunits. The catalytic subunit is a type 1phosphatase � isoform (PP1c�) and the larger regulatory sub-unit, MYPT1, is critical for regulation and localization, whilethe function of the smaller regulatory subunit remains unclear(for a review, see Ref. 22). In the last 15 years, the regulationof MLCP activity has become increasingly more appreciated interms of regulating smooth muscle contraction and Ca2� sen-sitivity (37). Smooth muscle Ca2� sensitization primarily oc-curs through two mechanisms involving the phosphorylation ofdistinct residues on MYPT1 or CPI-17 (37), which leads to theinhibition of MLCP. CPI-17 is a potent and selective inhibitorof MLCP and its inhibitory influence is greatly increased uponphosphorylation of its Thr38 residue (34). CPI-17 is considereda point of convergence since numerous signaling pathways,including PKC (7, 23), ROK (7, 23), and ILK (5), have beenshown to induce smooth muscle contraction and Ca2� sensiti-zation via CPI-17-dependent inhibition of MLCP. Interest-ingly, Kitazawa et al. (26) reported that changes in Ca2�

sensitivity were associated with reductions in CPI-17 levels in�-escin and Triton X-100 skinned smooth muscle. Further-more, reconstitution of CPI-17 and its potent activator, PKC�enhanced the Ca2� sensitivity in Triton X-100-skinned smoothmuscle (26). Woodsome et al. (44) reported that expression ofCPI-17 correlates with Ca2� sensitization via PKC in tonic andphasic smooth muscle. Thus, our data showing a decrease inCa2� sensitivity following �-escin permeabilization of lym-phatic muscle in association with a loss of CPI-17 protein areconsistent with several reports in the literature. Future studiesare warranted to determine the signaling pathways that modulatelymphatic 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 ofpermeabilized phasic lymphatic muscle is different from thoseof tonic vascular smooth muscle. We postulate that the funda-mental differences in myofilament activation between thesetissues contribute to their specialized functions in regulatingblood pressure (arteries) and volume (veins) and in the gener-ation and regulation of lymph flow (lymphatics). In addition,we provide indirect evidence that CPI-17-dependent inhibitionof MLCP is an important regulatory mechanism of lymphaticmuscle contraction and Ca2� sensitivity. The use of thesepermeabilized lymphatic muscle preparations will accelerateour understanding of the unique contractile properties of thistissue. Furthermore, these types of experiments will allow us todetermine whether alterations in the lymphatic myofilamentcontribute to impaired function of the lymph pump in diseaseconditions such as chronic edema.

ACKNOWLEDGMENTS

The authors would like to thank Scott Zawieja for his help in preparingcalcium solutions.

GRANTS

This study was supported by grants from National Institutes of HealthHL-080526 and KO2 HL086650 to M. Muthuchamy, and HL-75199 toD. Zawieja.



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calcium sensitivity and cooperativity of permeabilized rat mesenteric lymphatics

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