Rat Mesenteric Vessels Research Articles

Mechanisms of Acute Alcohol Intoxication-Induced Modulation of Cyclic Mobilization of [Ca²⁺] in Rat Mesenteric Lymphatic Vessels.

We have demonstrated that acute alcohol intoxication (AAI) increases the magnitude of Ca(2+) transients in pumping lymphatic vessels. We tested the contribution of extracellular Ca(2+) via L-type Ca(2+) channels and intracellular Ca(2+) release from the sarcoplasmic reticulum (SR) to the AAI-induced increase in Ca(2+) transients. AAI was produced by intragastric administration of 30% alcohol to conscious, unrestrained rats; isovolumic administration of water served as the control. Mesenteric lymphatic vessels were isolated, cannulated, and loaded with Fura-2 AM to measure changes in intracellular Ca(2+). Measurements were made at intraluminal pressures of 2, 6, and 10 cm H2O. L-type Ca(2+) channels were blocked with nifedipine; IP-3 receptors were inhibited with xestospongin C; and SR Ca(2+) release and Ca(2+) pool (Ca(2+) free APSS) were achieved using caffeine. Nifedipine reduced lymphatic Ca(2+) transient magnitude in both AAI and control groups at all pressures tested, but reduced lymphatic contraction frequency only in the control group. Xestospongin C did not significantly change any of the Ca(2+) parameters in either group; however, fractional shortening increased in the controls at low transmural pressure. RyR (ryanodine receptor) activation with caffeine resulted in a single contraction with a greater Ca(2+) transient in lymphatics from AAI than those from controls. SR Ca(2+) pool was also greater in lymphatics isolated from AAI- than from control animals. These data suggest that 1) L-type Ca(2+) channels contribute to the AAI-induced increase in lymphatic Ca(2+) transient, 2) blockage of IP-3 receptors could increase calcium sensitivity, and 3) AAI increases Ca(2+) storage in the SR in lymphatic vessels.

Involvement of TRPM4 in Stretch‐Induced Modulation of Lymphatic Pumping

Rhythmic and phasic contractions of collecting lymphatic vessels are critically involved in tissue fluid homeostasis and immune response. This contractile activity, or lymphatic pumping, is exquisitely regulated by luminal fluid pressure. However, the molecular contributors of stretch‐induced lymphatic pumping are not known. Some members of the TRP channel family, in particular TRPM4,have been shown to respond to stretch or pressure and to contribute to myogenic vasoconstriction and vascular response to stretch.Using pressure myography, electrophysiology, pharmacology and quantitative real‐time PCR, we tested the hypothesis that TRPM4 is also a major contributor of the lymphatic vessel response to stretch.We demonstrated the expression of TRPM4 mRNA in rat mesenteric lymphatic vessels and showed that the contraction frequency of the vessels pressurized at 3 mmHg was inhibited in a concentration‐dependent manner by the TRPM4 blocker 9‐phenanthrol (IC50=1.2 µM). The blocker (10 µM) also inhibited the pressure‐induce increase in contraction frequency in an endothelium‐independent manner and caused a hyperpolarization of the lymphatic muscle. These findings suggest an important role for TRPM4 in the ability of lymphatic vessels to adapt their contractile activity to changes in intraluminal lymph pressure.

TNF‐α Inhibits Rat Mesenteric Lymphatic Pumping via Activation of the NF‐κB and Upregulation of iNOS

Upregulated TNF‐α production and dysfunctional lymphatic pumping are observed in inflammatory bowel disease pathogenesis. It has been previously demonstrated that TNF‐α could decrease arterial contractility via activation of the inducible nitric oxide synthase (iNOS) and that iNOS is involved in impaired mesenteric lymphatic pumping in animal model of intestinal inflammation. We investigated the effects of TNF‐α on rat mesenteric lymphatic pumping and hypothesized that the cytokine may impair lymphatic contractile function during intestinal inflammation by upregulating the synthesis of iNOS and thus the production of nitric oxide.Using pressure myography, we examined the contractile function of isolated rat mesenteric lymphatic vessels following a 24‐hr treatment with TNF‐α. We observed that contraction frequency was significantly and concentration‐dependently decreased by TNF‐α, an effect restored following administration of inhibitors of NF‐κB (PDTC), iNOS (1400W), guanylate synthase (ODQ) and ATP‐sensitive K+ channels (glibenclamide), but not the cyclooxygenase inhibitor, indomethacin. The involvements of NF‐κB and iNOS were further confirmed by western blot analysis and quantitative real‐time PCR, which revealed upregulations of phospho‐IκB and iNOS mRNA in TNF‐α‐treated vessels that were minimized in the presence of PDTC.These findings suggest that TNF‐α decreases lymphatic contractility via activation of the NF‐κB‐iNOS pathway.

Phosphate as a cardiovascular risk factor: effects on vascular and endothelial function

BackgroundHyperphosphataemia is a risk factor for accelerated cardiovascular disease in chronic kidney disease. The mechanism is poorly understood; it is unclear whether phosphate has direct effects or effects mediated via calcification or FGF23. We investigated direct effects of phosphate on endothelial function using myography to study rat and human blood vessels. In addition we assessed the effects of phosphate loading on endothelial function in a clinical study. MethodsResistance vessels from patients with (n=12) and without (n=13) chronic kidney disease were incubated in normal or high phosphate. Vasoconstrictor and vasorelaxation responses were measured. Concentration-response curves were constructed and comparisons made. Identical experiments were performed in rat mesenteric vessels with and without phosphodiesterase type 5 inhibitor. A cross-over study was done in 19 healthy volunteers receiving phosphate supplements or binders and endothelial function measured by flow mediated dilatation (FMD). Primary outcome was percent change in FMD from baseline. FindingsNine to 13 vessels were used in each group. Endothelium-dependent vasodilatation was impaired in high compared with normal phosphate in rat (mean maximum vasodilatation 64% [SE 9] vs 95 [1], p<0·001) and human vessels with (25·3 [11·1] vs 75·7 [13·6], p<0·001) and without chronic kidney disease (42·9 [12] vs 79·4 [8·2], p=0·003). In rat vessels, these effects were reversed by a phosphodiesterase type 5 inhibitor. In vivo in volunteers, endothelial function was reduced by phosphate loading (median maximum vasodilatation 3·38% [IQR 2·57–5·26] vs 8·4 [6·2–11·6], p<0·001); this effect was independent of serum phosphate concentration but associated with urinary phosphate excretion and serum FGF23 concentrations. InterpretationProlonged exposure to phosphate is associated with endothelial dysfunction, a direct effect of phosphate, which might contribute to cardiovascular risk in chronic kidney disease. In a high phosphate environment, endothelial and vascular dysfunction is evident in blood vessels and in man exposed to prolonged oral phosphate loading. These effects might be mediated by disruption of the NO pathway. FundingBritish Heart Foundation, Darlinda's Charity for Renal Research.

Distinct roles of L- and T-type voltage-dependent Ca2+ channels in regulation of lymphatic vessel contractile activity.

Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of collecting lymphatic vessels through which lymph is propelled back into the blood circulation. This rhythmic contractile activity (i.e. lymphatic pumping) increases in rate with increase in luminal pressure and relies on activation of nifedipine-sensitive voltage-dependent Ca(2+) channels (VDCCs). Despite their importance, these channels have not been characterized in lymphatic vessels. We used pressure- and wire-myography as well as intracellular microelectrode electrophysiology to characterize the pharmacological and electrophysiological properties of L-type and T-type VDCCs in rat mesenteric lymphatic vessels and evaluated their particular role in the regulation of lymphatic pumping by stretch. We complemented our study with PCR and confocal immunofluorescence imaging to investigate the expression and localization of these channels in lymphatic vessels. Our data suggest a delineating role of VDCCs in stretch-induced lymphatic vessel contractions, as the stretch-induced increase in force of lymphatic vessel contractions was significantly attenuated in the presence of L-type VDCC blockers nifedipine and diltiazem, while the stretch-induced increase in contraction frequency was significantly decreased by the T-type VDCC blockers mibefradil and nickel. The latter effect was correlated with a hyperpolarization. We propose that activation of T-type VDCCs depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L-type VDCCs, which drive the strength of contractions.

The position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo.

Until now, there has been no tool available to provide lymphatic researchers the ability to perform experiments in tissue explants containing lymphatic vessels under tissue position- and lymphatic lumen-controlled conditions. In this article we provide technical details and description of the method of using the newly developed and implemented the position- and lymphatic lumen-controlled tissue chambers to study live lymphatic vessels and surrounding tissues ex vivo. In this study, we, for the first time, performed detailed comparative analysis of the contractile and pumping activity of rat mesenteric lymphatic vessels (MLVs) situated within tissue explants mounted in new tissue chambers and isolated, cannulated, and pressurized rat MLVs maintained in isolated vessel setups. We found no significant differences of the effects of both transmural pressure- and wall shear stress sensitivities of MLVs in tissue chambers and isolated MLVs. We conclude that this new experimental tool, a position- and lymphatic lumen-controlled tissue chamber, allows precise investigation of lymphatic function of MLVs interacting with elements of the tissue microenvironment. This method provides an important new set of experimental tools to investigate lymphatic function.

Electrophysiological properties of rat mesenteric lymphatic vessels and their regulation by stretch.

In mammals, lymph is propelled centrally primarily via the phasic contractions of collecting lymphatic vessels, known as lymphatic pumping. Electrophysiological studies conducted in guinea pig and sheep mesenteric lymphatic vessels indicate that contractions are initiated in the lymphatic muscle by nifedipine-sensitive action potentials (APs). Lymphatic pumping is highly sensitive to luminal fluid loading and the mechanical properties of this stretch-induced pumping have been consistently studied, in particular in rat mesenteric lymphatic vessels. However, membrane potential (Vm) and the electrophysiological events underlying stretch-induced lymphatic pumping have not been investigated in the rat. The aim of this study was thus to examine the properties of rat mesenteric lymphatic muscle Vm under resting conditions and to assess changes in Vm caused by distension. Lymphatic muscle Vm was measured with sharp intracellular microelectrodes either in unstretched conditions or under isometric tension provided by a wire-myograph. In unstretched vessels, Vm was -48 ± 2 mV (n=30). APs (amplitude ∼25 mV) were observed at a frequency of ∼8/min and were abolished by nifedipine. Under isometric tension, Vm was less polarized (-36 ± 1 mV, n=23), even at minimum tension. Increase in tension led to increase in contraction strength and contraction/AP frequency, while Vm was slightly hyperpolarized and AP amplitude not markedly altered. In our experimental conditions, rat lymphatic muscle has electrophysiological characteristics similar to that in other species. It responds to an increase in isometric tension with an increase in AP frequency, but resting Vm is not significantly affected.

Rho Kinase Enhances Contractions of Rat Mesenteric Collecting Lymphatics

The mechanisms that control phasic and tonic contractions of lymphatic vessels are poorly understood. We hypothesized that rho kinase ROCK, previously shown to increase calcium (Ca2+) sensitivity in vascular smooth muscle, enhances lymphatic contractile activity in a similar fashion. Contractions of isolated rat mesenteric lymphatic vessels were observed at a luminal pressure of 2 cm H2O in a 37°C bath. The expression of ROCK in isolated rat mesenteric lymphatic vessels was assessed by Western blotting and confocal microscopy. The role of ROCK in contractile function was tested using two specific yet structurally distinct inhibitors: H1152 (0.1–10 μM) and Y-27632 (0.5–50 μM). In addition, lymphatics were transfected with constitutively active (ca)-ROCK protein (2 μg/ml) to assess gain of contractile function. Vessel diameter and the concentration of intracellular free Ca2+ ([Ca2+]i) were simultaneously measured in a subset of isolated lymphatics loaded with the Ca2+-sensing dye fura-2. The results show expression of both the ROCK1 and ROCK2 isoforms in lymphatic vessels. Inhibition of ROCK increased lymphatic end diastolic diameter and end systolic diameter in a concentration-dependent manner. Significant reductions in lymphatic tone and contraction amplitude were observed after treatment 1–10 μM H1152 or 25–50 μM Y-27632. H1152 (10 μM) also significantly reduced contraction frequency. Transient increases in [Ca2+]i preceded each phasic contraction, however this pattern was disrupted by either 10 μM H1152 or 50 μM Y-27632 in the majority of lymphatics studied. The significant decrease in tone caused by H1152 or Y-27632 was not associated with a significant change in the basal [Ca2+]i between transients. Transfection with ca-ROCK protein enhanced lymphatic tone, but was not associated with a significant change in basal [Ca2+]i. Our data suggest that ROCK mediates normal tonic constriction and influences phasic contractions in lymphatics. We propose that ROCK modulates Ca2+ sensitivity of contractile proteins in lymphatics.

Doxorubicin inhibits lymphatic function in vitro and in vivo (655.1)

Doxorubicin (DOX) is an independent risk factor for lymphedema in breast cancer patients after surgery and/or radiation therapy. The mechanisms by which DOX contributes to lymphedema are unclear. We hypothesized that DOX directly inhibits lymphatic contractions and lymph flow by a mechanism distinct from its cytotoxic action. Isolated rat mesenteric lymph vessels (LVs) were cannulated, pressurized (4‐5 mm Hg) and equilibrated in a no‐flow system using physiological salt solution (37°C) until spontaneous contractions were stable. An edge‐detection system recorded changes in external diameter. Adding DOX into the bath at increasing concentrations (0.5 to 20 µmol/L; n=7) progressively attenuated the amplitude and area under the curve (AUC) of LV contractions, culminating in reductions of ~55% (amplitude) and ~60% (AUC). Washout of DOX partially restored contractile activity. High‐resolution, intravital optical imaging of rat mesenteric LVs in vivo also revealed that DOX (10 µmol/L) inhibits LV spontaneous contractions and reduces lymph flow (n=2). DOX reduced lymph flow by ~66% (t=10 min) and 100% (t=30 min) from baseline. Thus, DOX may contribute to lymphedema by directly inhibiting LV contractile function as an off‐target effect.Grant Funding Source: Partially supported by a Rho Chi‐AFPE Graduate Fellowship (AS)

Efavirenz induces interactions between leucocytes and endothelium through the activation of Mac-1 and gp150,95

The potential cardiovascular (CV) toxicity associated with combined antiretroviral therapy (cART) has been attributed mainly to the nucleoside reverse transcriptase inhibitors abacavir and didanosine. However, the other two components of cART--non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs)--may also be implicated, either directly or by influencing the action of the other drugs. This study evaluates the acute direct effects of the NNRTIs efavirenz and nevirapine and one of the most widely employed PIs, lopinavir, on leucocyte-endothelium interactions, a hallmark of CV disease. Drugs were analysed in vitro in human cells (interactions of peripheral blood polymorphonuclear or mononuclear cells with human umbilical vein endothelial cells) using a flow chamber system, and in vivo in rat mesenteric vessels by means of intravital microscopy. The expression of adhesion molecules in leucocytes and endothelial cells was studied by flow cytometry, and the role of these molecules in white cell recruitment was evaluated by pre-treating human cells or rats with blocking antibodies. Efavirenz and nevirapine, but not lopinavir, increased the rolling flux and adhesion of leucocytes in vitro and in vivo while inducing emigration in rat venules. Efavirenz, but not nevirapine, augmented the levels of CD11b, CD11c and CD18 in neutrophils and monocytes. The actions of efavirenz, but not of nevirapine, were reversed by antibodies against Mac-1 (CD11b/CD18), gp150,95 (CD11c/CD18) or ICAM-1 (CD54). NNRTIs, but not PIs, interfere with leucocyte-endothelial interactions. However, differences between efavirenz and nevirapine suggest a specific CV profile for each compound.

Profile of Leukocyte-Endothelial Cell Interactions Induced in Venules and Arterioles by Nucleoside Reverse-Transcriptase Inhibitors In Vivo

There is controversy regarding cardiovascular (CV) toxicity of the nucleoside reverse-transcriptase inhibitors used to treat human immunodeficiency virus infection. We evaluated the effects of nucleoside reverse-transcriptase inhibitors on leukocyte-endothelium interactions, a hallmark of CV diseases, in rat mesenteric vessels using intravital microscopy and in human arterial cells using a flow chamber system. Abacavir and didanosine increased rolling, adhesion and emigration in rat vessels. These effects were reversed with antibodies against Macrophage-1 antigen (Mac-1) or intercellular adhesion molecule 1 and were reproduced in human cells. Lamivudine, zidovudine, emtricitabine, and tenofovir had no effects. Our results support the association of abacavir and didanosine with CV diseases.

Development of a model of a multi-lymphangion lymphatic vessel incorporating realistic and measured parameter values

Our published model of a lymphatic vessel consisting of multiple actively contracting segments between non-return valves has been further developed by the incorporation of properties derived from observations and measurements of rat mesenteric vessels. These included (1) a refractory period between contractions, (2) a highly nonlinear form for the passive part of the pressure-diameter relationship, (3) hysteretic and transmural-pressure-dependent valve opening and closing pressure thresholds and (4) dependence of active tension on muscle length as reflected in local diameter. Experimentally, lymphatic valves are known to be biased to stay open. In consequence, in the improved model, vessel pumping of fluid suffers losses by regurgitation, and valve closure is dependent on backflow first causing an adverse valve pressure drop sufficient to reach the closure threshold. The assumed resistance of an open valve therefore becomes a critical parameter, and experiments to measure this quantity are reported here. However, incorporating this parameter value, along with other parameter values based on existing measurements, led to ineffective pumping. It is argued that the published measurements of valve-closing pressure threshold overestimate this quantity owing to neglect of micro-pipette resistance. An estimate is made of the extent of the possible resulting error. Correcting by this amount, the pumping performance is improved, but still very inefficient unless the open-valve resistance is also increased beyond the measured level. Arguments are given as to why this is justified, and other areas where experimental data are lacking are identified. The model is capable of future adaptation as new experimental data appear.

Histamine is novel endothelium‐derived relaxing factor in rat mesenteric lymphatic vessels

Our recent studies (Nagai et al., 2011) suggested the existence of yet undiscovered, shear‐dependent, but NO‐independent regulatory mechanism in rat mesenteric lymphatic vessels (MLV). In this study we performed for the first time the detailed evaluation of potential involvement of histamine in such regulation of MLV contractility in F‐344 rats of various age groups (3‐mo, 9‐mo and 24‐mo). Using confocal and epifluorescent IHC labeling, we confirmed the presence of histidine decarboxylase (HDC), histamine‐producing enzyme, within the lymphatic endothelial cells in MLV. These results were re‐confirmed by Western blot analysis. Using Vivo‐Morpholino technology we were able to knock‐down effectively HDC protein within MLV. In experiments with isolated MLV, we found that only combined blockade of eNOS (NO production) by L‐NAME and HDC (histamine production) by α‐methyl‐DL‐histidine dihydrochloride was able to completely eliminate all shear‐dependency of lymphatic contractile self‐adjustment during periods of steady increases of imposed flow. Such regulatory role of histamine appears to be not severely altered by aging. Up‐to‐date results shows that histamine is not involved in fast phasic contraction‐generated shear‐dependent contractile regulation. Our data provide ground for new understanding on the role of histamine in regulation of lymphatic function. NIH R01 AG030578, HL094269.

Role of voltage‐dependent calcium channels in stretch‐induced lymphatic vessel contractions

Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of lymphatic vessels, which increase in rate with increase in luminal pressure. The contractions depend on activation of voltage‐dependent calcium channel (VDCC), which have not been characterized in lymphatic vessels. We used pressure‐ and wire‐myography, electrophysiology, PCR and immunofluorescence imaging to investigate the electrophysiological properties of L‐type and T‐type VDCCs and their role on stretch‐induced lymphatic contractions.Members of the VDCC family were expressed at the messenger RNA and protein level in rat mesenteric lymphatic vessels. The stretch‐induced increase in force was significantly attenuated in the presence of L‐type VDCC blockers nifedipine and diltiazem, while the T‐type VDCC blockers mibefradil and nickel significantly decreased the stretch‐induced increase in contraction frequency. Furthermore, nifedipine and diltiazem depolarized, while mibefradil and nickel hyperpolarized lymphatic muscle membrane potential.Our data suggest a delineating role of VDCCs in stretch‐induced lymphatic vessel contractions. We propose that activation of T‐type VDCC depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L‐type VDCCs, which drive the strength of contractions.Supported by NIH

Muscle cells in rat mesenteric lymphatic vessels of various ages

The detailed quantitative evaluation of potential aging‐associated changes in muscle cell density in mesenteric lymphatic vessels (MLV) has never been performed. In this study we, for the first time, performed detailed evaluation of muscle cell density in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3‐mo, 9‐mo and 24‐mo Fischer‐344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream and above lymphatic valves possess the lowest density of lymphatic muscle cells. Most of the high muscle cell density zones exist downstream to the lymphatic valve. The muscle cell density of these zones is not affected by aging while pre‐valve and valve zones demonstrate significant aging‐associated decrease in muscle cell density. The low muscle cell density zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned above and near lymphatic valve and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging‐associated changes may delimit adaptive reserves of aged lymphatic vessels. NIH R01 AG030578, HL094269.

Lymphatic Muscle Cells in Rat Mesenteric Lymphatic Vessels of Various Ages

Recent studies on aging-associated changes in mesenteric lymph flow in situ demonstrated predominance of the severe negative chronotropic effect of aging on the contractility of aged mesenteric lymphatic vessels (MLV). At the same time, contraction amplitude of the aged vessels was only slightly diminished by aging and can be rapidly stimulated within 5-15 minutes. However, the detailed quantitative evaluation of potential aging-associated changes in muscle cells investiture in MLV has never been performed. In this study we, for the first time, performed detailed evaluation of muscle cells investiture in MLV in reference to the position of lymphatic valve in different zones of lymphangion within various age groups (3-mo, 9-mo and 24-mo Fischer-344 rats). Using visual and quantitative analyses of the images of MLV immunohistochemically labeled for actin, we confirmed that the zones located close upstream (pre-valve zones) and above lymphatic valves (valve zones) possess the lowest investiture of lymphatic muscle cells. Most of the high muscle cells investiture zones exist downstream to the lymphatic valve (post-valve zones). The muscle cells investiture of these zones is not affected by aging, while pre-valve and valve zones demonstrate significant aging-associated decrease in muscle cells investiture. The low muscle cells investiture zones in lymphatic vessels consist of predominantly longitudinally oriented muscle cells which are positioned in pre-valve and valve zones and connect adjacent lymphangions. These cells may provide important functional impact on the biomechanics of the lymphatic valve gating and electrical coupling between lymphangions, while their aging-associated changes may delimit adaptive reserves of aged lymphatic vessels.

Hybrid PIV–PTV technique for measuring blood flow in rat mesenteric vessels

The micro-particle tracking velocimetry (μ-PTV) technique is used to obtain the velocity fields of blood flow in the microvasculature under in vivo conditions because it can provide the blood velocity distribution in microvessels with high spatial resolution. The in vivo μ-PTV technique usually requires a few to tens of seconds to obtain a whole velocity profile across the vessel diameter because of the limited number density of tracer particles under in vivo conditions. Thus, the μ-PTV technique alone is limited in measuring unsteady blood flows that fluctuate irregularly due to the heart beating and muscle movement in surrounding tissues. In this study, a new hybrid PIV–PTV technique was established by combining PTV and particle image velocimetry (PIV) techniques to resolve the drawbacks of the μ-PTV method in measuring blood flow in microvessels under in vivo conditions. Images of red blood cells (RBCs) and fluorescent particles in rat mesenteric vessels were obtained simultaneously. Temporal variations of the centerline blood velocity were monitored using a fast Fourier transform-based cross-correlation PIV method. The fluorescence particle images were analyzed using the μ-PTV technique to extract the spatial distribution of the velocity vectors. Data from the μ-PTV and PIV methods were combined to obtain a better estimate of the velocity profile in actual blood flow. This technique will be useful in investigating hemodynamics in microcirculation by measuring unsteady irregular blood flows more accurately.

Ca2+‐related proteins associated with intracellular stores in rat lymphatics

Calcium plays essential roles in the contraction of striated and smooth muscle types. Changes in cytoplasmic concentration of Ca2+ depend in general on the calcium channels and pumps at the cytoplasmic and endoplasmic/sarcoplasmic reticulum membrane. As we have shown previously, lymphatic muscle has a unique combination of the striated and smooth muscle contractile and regulatory proteins (Muthuchamy et al., 2003). However, very little is known about the composition of the Ca2+ ‐channels and pumping structures in the intracellular stores of lymphatic muscle. Our immunohistochemistry data with confocal microcopy from whole isolated vessels preparation, frozen sections and lymphatic muscle cell culture demonstrates the presence of inositol 1,4,5‐triphosphate receptor type 3 (IP3R3) channels, Ca2+ transporting ATPase type 2a1 (SERCA2a1) and Ca2+ transporting ATPase type 2a2 (SERCA2a2) at intracellular stores of rat mesenteric lymphatic vessels, cervical lymphatics and thoracic duct.Supported by NIH HL096552

Regional Heterogeneity of Length–Tension Relationships in Rat Lymph Vessels

Heterogeneity of the length-tension relationships in lymph vessels has never been evaluated systematically. In this study we measured the length-tension relationships in lymph vessels from three different regions of the rat: thoracic duct, cervical, and femoral lymph vessels, and compared the results to our previous measurements of rat mesenteric lymph vessels. We performed isometric force measurements on activated and passive lymph vessel segments using a small-vessel wire myograph. We found that all groups of vessels had relatively broad plateaus in their active tension versus length relationships, suggesting that they are adapted to generate near-maximal tensions over a relatively wide range of preloads (at least 0.85-1.05 L(0)). Thoracic duct exhibited the flattest active tension curve, particularly for peak active tension, in which there was less than a 5% change in peak active tension from 0.75 to 1.30 of optimal length. Femoral lymph vessels were able to withstand the highest estimated pressures, followed by mesenteric and cervical vessels and then thoracic duct. We conclude that lymph vessels effectively adapt their contractile force to the particular hydrodynamic conditions (transmural pressures and intraluminal flows) that exist in different regions of the lymphatic system.

Aging-Associated Alterations in Contractility of Rat Mesenteric Lymphatic Vessels

To evaluate the age-related changes in pumping of mesenteric lymphatic vessels in 9- and 24-month-old male Fisher-344 rats. Lymphatic diameters, contraction amplitude, contraction frequency, and fractional pump flow were determined in isolated MLV before and after l-NAME application. The data demonstrate a severe weakening of the lymphatic pump in aged MLV including diminished lymphatic contraction amplitude, contraction frequency, and as a result, lymphatic pump activity. The data also suggest that the imposed flow gradient-generated shear-dependent relaxation does not exist in aged rat MLV, and the sensitivity of both adult and aged MLV to such shear cannot be eliminated by nitric oxide (NO) synthases blockade. These data provide new evidence of lymphatic regional heterogeneity for both adult and aged MLV. In MLV, a constant interplay between the tonic and phasic components of the myogenic response and the shear-dependent release of NO predominantly determine the level of contractile activity; the existence of another shear-dependent, but NO-independent regulatory mechanism is probably present. Aging remarkably weakens MLV contractility, which would predispose this lymphatic network to lower total lymph flow in resting conditions and limit the ability to respond to an edemagenic challenge in the elderly.