Quantification of the Whole Lymph Node Vasculature Based on Tomography of the Vessel Corrosion Casts.

Lymph nodes (LN) are crucial for immune function, and comprise an important interface between the blood and lymphatic systems. Blood vessels (BV) in LN are highly specialized, featuring high endothelial venules across which most of the resident lymphocytes crossed. Previous measurements of overall lymph and BV flow rates demonstrated that fluid also crosses BV walls, and that this is important for immune function. However, the spatial distribution of the BV in LN has not been quantified to the degree necessary to analyse the distribution of transmural fluid movement. In this study, we seek to quantify the spatial localization of LNBV, and to predict fluid movement across BV walls. MicroCT imaging of murine popliteal LN showed that capillaries were responsible for approximately 75% of the BV wall surface area, and that this was mostly distributed around the periphery of the node. We then modelled blood flow through the BV to obtain spatially resolved hydrostatic pressures, which were then combined with Starling's law to predict transmural flow. Much of the total 10 nL/min transmural flow (under normal conditions) was concentrated in the periphery, corresponding closely with surface area distribution. These results provide important insights into the inner workings of LN, and provide a basis for further exploration of the role of LN flow patterns in normal and pathological functions.

Acute exposure to space flight results in evidence of reduced lymph Transport, tissue fluid Shifts, and immune alterations in the rat gastrointestinal system.

Space flight causes a number of alterations in physiological systems, changes in the immunological status of subjects, and altered interactions of the host to environmental stimuli. We studied the effect of space flight on the lymphatic system of the gastrointestinal tract which is responsible for lipid transport and immune surveillance which includes the host interaction with the gut microbiome. We found that there were signs of tissue damage present in the space flown animals that was lacking in ground controls (epithelial damage, crypt morphological changes, etc.). Additionally, morphology of the lymphatic vessels in the tissue suggested a collapsed state at time of harvest and there was a profound change in the retention of lipid in the villi of the ileum. Contrary to our assumptions there was a reduction in tissue fluid volume likely associated with other fluid shifts described. The reduction of tissue fluid volume in the colon and ileum is a likely contributing factor to the state of the lymphatic vessels and lipid transport issues observed. There were also associated changes in the number of MHC-II+ immune cells in the colon tissue, which along with reduced lymphatic competence would favor immune dysfunction in the tissue. These findings help expand our understanding of the effects of space flight on various organ systems. It also points out potential issues that have not been closely examined and have to potential for the need of countermeasure development.

IL-1ß reduces tonic contraction of mesenteric lymphatic muscle cells, with the involvement of cycloxygenase-2 and prostaglandin E2.

BACKGROUND AND PURPOSE: The lymphatic system maintains tissue homeostasis by unidirectional lymph flow, maintained by tonic and phasic contractions within subunits, 'lymphangions'. Here we have studied the effects of the inflammatory cytokine IL-1ß on tonic contraction of rat mesenteric lymphatic muscle cells (RMLMC). EXPERIMENTAL APPROACH: We measured IL-1ß in colon-conditioned media (CM) from acute (AC-CM, dextran sodium sulfate) and chronic (CC-CM, T-cell transfer) colitis-induced mice and corresponding controls (Con-AC/CC-CM). We examined tonic contractility of RMLMC in response to CM, the cytokines h-IL-1ß or h-TNF-α (5, 10, 20 ng·mL(-1) ), with or without COX inhibitors [TFAP (10(-5) M), diclofenac (0.2 × 10(-5) M)], PGE2 (10(-5) M)], IL-1-receptor antagonist, Anakinra (5 µg·mL(-1) ), or a selective prostanoid EP4 receptor antagonist, GW627368X (10(-6) and 10(-7) M). KEY RESULTS: Tonic contractility of RMLMC was reduced by AC- and CC-CM compared with corresponding control culture media, Con-AC/CC-CM. IL-1ß or TNF-α was not found in Con-AC/CC-CM, but detected in AC- and CC-CM. h-IL-1ß concentration-dependently decreased RMLMC contractility, whereas h-TNF-α showed no effect. Anakinra blocked h-IL-1ß-induced RMLMC relaxation, and with AC-CM, restored contractility to RMLMC. IL-1ß increased COX-2 protein and PGE2 production in RMLMC.. PGE2 induced relaxations in RMLMC, comparable to h-IL-1ß. Conversely, COX-2 and EP4 receptor inhibition reversed relaxation induced by IL-1ß. CONCLUSIONS AND IMPLICATIONS: The IL-1ß-induced decrease in RMLMC tonic contraction was COX-2 dependent, and mediated by PGE2 . In experimental colitis, IL-1ß and tonic lymphatic contractility were causally related, as this cytokine was critical for the relaxation induced by AC-CM and pharmacological blockade of IL-1ß restored tonic contraction.

Measurement of shear stress-mediated intracellular calcium dynamics in human dermal lymphatic endothelial cells.

The shear stress applied to lymphatic endothelial cells (LEC) by lymph flow changes dramatically under normal conditions as well as in response to disease conditions and immune reactions. In general, LEC are known to regulate the contraction frequency and strength of lymphatic pumping in response to shear stress. Intracellular calcium concentration ([Ca(2+)]i) is an important factor that regulates lymphatic contraction characteristics. In this study, we measured changes in the [Ca(2+)]i under different shear stress levels and determined the source of this calcium signal. Briefly, human dermal LEC were cultured in custom-made microchannels for 3 days before loading with 2 µM fura-2 AM, a ratiometric calcium dye to measure [Ca(2+)]i. Step changes in shear stress resulted in a rapid increase in [Ca(2+)]i followed by a gradual return to the basal level and sometimes below the initial baseline (45.2 ± 2.2 nM). The [Ca(2+)]i reached a peak at 126.2 ± 5.6 nM for 10 dyn/cm(2) stimulus, whereas the peak was only 71.8 ± 5.4 nM for 1 dyn/cm(2) stimulus, indicating that the calcium signal depends on the magnitude of shear stress. Removal of the extracellular calcium from the buffer or pharmocological blockade of calcium release-activated calcium (CRAC) channels significantly reduced the peak [Ca(2+)]i, demonstrating a role of extracellular calcium entry. Inhibition of endoplasmic reticulum (ER) calcium pumps showed the importance of intracellular calcium stores in the initiation of this signal. In conclusion, we demonstrated that the shear-mediated calcium signal is dependent on the magnitude of the shear and involves ER store calcium release and extracellular calcium entry.

Increased susceptibility of aging kidney to ischemic injury: identification of candidate genes changed during aging, but corrected by caloric restriction.

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism underlying the increased susceptibility to injury remains undefined. These experiments were designed to investigate the influence of age on the response of the kidney to ischemic injury and to identify candidate genes that may mediate this response. Renal slices prepared from young (5 mo), aged ad libitum (aged-AL; 24 mo), and aged caloric-restricted (aged-CR; 24 mo) male Fischer 344 rats were subjected to ischemic stress (100% N(2)) for 0-60 min. As assessed by biochemical and histological evaluation, slices from aged-AL rats were more susceptible to injury than young counterparts. Importantly, caloric restriction attenuated the increased susceptibility to injury. In an attempt to identify the molecular pathway(s) underlying this response, microarray analysis was performed on tissue harvested from the same animals used for the viability experiments. RNA was isolated and the corresponding cDNA was hybridized to CodeLink Rat Whole Genome Bioarray slides. Subsequent gene expression analysis was performed using GeneSpring software. Using two-sample t-tests and a twofold cut-off, the expression of 92 genes was changed during aging and attenuated by caloric restriction, including claudin-7, kidney injury molecule-1 (Kim-1), and matrix metalloproteinase-7 (MMP-7). Claudin-7 gene expression peaked at 18 mo; however, increased protein expression in certain tubular epithelial cells was seen at 24 mo. Kim-1 gene expression was not elevated at 8 or 12 mo but was at 18 and 24 mo. However, changes in Kim-1 protein expression were only seen at 24 mo and corresponded to increased urinary levels. Importantly, these changes were attenuated by caloric restriction. MMP-7 gene expression was decreased at 8 mo, but an age-dependent increase was seen at 24 mo. Increased MMP-7 protein expression in tubular epithelial cells at 24 mo was correlated with the gene expression pattern. In summary, we identified genes changed by aging and changes attenuated by caloric restriction. This will facilitate investigation into the molecular mechanism mediating the age-related increase in susceptibility to injury.

Relationship between cardiac protein tyrosine phosphorylation and myofibrillogenesis during axolotl heart development.

The axolotl, Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of protein tyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the state of protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomeric myofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosine phosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40-41 showed less tyrosine phosphorylated staining as compared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower content of phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstrated that P-Tyr staining gradually increased being localized primarily at cell-cell boundaries and cell-extracellular matrix boundaries. In contrast, mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell-cell and cell-matrix junctions. We also delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resulted in a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation is critical during myofibrillogenesis and embryonic heart development in axolotls.

Development and characterization of endothelial cells from rat microlymphatics.

BACKGROUND: The lymphatic endothelium is important to the functioning of the lymphatic system, including lymphatic remodeling, control of vessel tone, and lymphatic movement of fluids, macromolecules, and cells. Many of these events occur principally at the level of the microlymphatics. To evaluate the role of the microlymphatic endothelium, a suitable cultured cell line would be useful. We have developed a technique to isolate and culture endothelial cells from microscopic lymphatics, approximately 100 microm in diameter. METHODS AND RESULTS: To isolate the rat mesenteric lymphatic endothelial cells (RMLEC), the rat was anesthetized and the mesentery carefully exteriorized. A suitable microlymphatic was located and carefully microdissected from the surrounding mesentery. The vessel was carefully cleaned, cannulated, everted, and then incubated on a gelatin-coated plastic culture dish until small patches of cells migrated off of the vessel (3-4 days later.) The explanted vessel was then removed. The remaining cells were cultured and screened for endothelial phenotype. Nonendothelial cells were destroyed. The endothelial nature of the remaining cells was verified by: 1) morphology, 2) uptake of fluorescent acetylated-LDL, 3) staining for von Wille-brand factor, PECAM-1, ecNOS, LYVE-1, VEGFR-3, and 4) essentially negative alpha-vascular smooth muscle actin staining. The defined RMLEC were passed and the profile of adhesion molecules present on the RMLEC was then determined using PCR and immunofluorescence. CONCLUSIONS: We developed and partially characterized a line of cultured microlymphatic endothelium. RMLEC express known endothelial- and lymphatic-specific markers as well as the following adhesion molecules: N-cadherin, E-cadherin, PECAM-1, alpha-catenin, beta-catenin, gamma-catenin, p120, and a variety of integrins.

Physiology of human lymphatic contractility: a historical perspective.

The lymphatic system is a transport system that has important roles in fluid/macromolecule homeostasis, lipid absorption, metastasis and immune function. It accomplishes these roles via the generation of a regulated lymph circulation which is dependent upon valves and pumps to overcome the normal fluid pressure gradients. Lymphatic contractility plays crucial roles in the regulation and generation of lymph transport. Whereas our understanding of lymphatic contractility in humans is somewhat limited, a number of studies both in situ and in vitro have provided important insights into the presence and modulation of lymphatic contractility. These studies have clearly demonstrated that lymphatic vessels from a number of different human tissues possess both tonic and phasic changes in contractility. These changes in contractility are presumably involved in the generation and regulation of lymph flow. It has been shown that human lymphatic contractility can be influenced by a number of neural and humoral agents as a means to control lymph transport. However our understanding of the physical and chemical factors which regulate both the spontaneous pumping activity and the vessel tone are more limited. An understanding of thefactors which regulate human lymph transport could provide valuable information on human biology that could be of benefit to the treatment and prevention of diseases.

Protein transfection of intact microvessels specifically modulates vasoreactivity and permeability.

Precise regulation of microvascular tone and barrier function is essential for proper coronary perfusion and performance. Agonist-induced alterations in either or both of these functions ultimately lead to microcirculatory dysfunction and cardiac insufficiency. Two important pathways involved in regulating vasomotor response and barrier function are the activation of nitric oxide synthase (NOS) and upregulation of protein kinase C (PKC). To date, studies of these two signaling proteins have relied mainly on pharmacological approaches. Unfortunately, the specificity of various inhibitors can be cause for concern. In order to address this problem, a protein transfection technique we developed for cultured endothelial cells has been modified and applied to isolated, intact coronary microvessels. Our results from green fluorescent protein transfection in arterioles and venules showed that this procedure could be used to introduce proteins into the microvascular wall. By transfecting inhibitor peptides against NOS and PKC into coronary arterioles and venules, we have been able to determine the specific roles of these two enzymes in vasodilation and hyperpermeability responses.

[Contractions of the lymphangion under low filling conditions and the absence of stretching stimuli. The possibility of the sucking effect]. / Sokrashcheniia limfangionov pri malom napolnenii i pri otsutstvii rastiagivaiushchikh stimulov. Vozmozhnost' prisasyvaiushchego éffekta.

Regulation of the function of the lymphatic pump is tightly connected to the degree of filling of the lymphangion. This mechanism is one of the principle ways that the lymphatics self-regulate lymph transport. But interpretation of the causes and consequences of this regulation is still far from perfect. The issue of whether automatism of electrical activity of the lymphatic smooth muscle cells or the distension of the cell membranes first causes depolarization, is a principal question which needs answering in order to understand the control of lymph transport. In our experiments with perfused isolated bovine mesenteric lymphangions, different values of lymph pressure were simulated. For this purpose, lowfrequency sinusoidal fluctuations of the input pressure were applied to lymphangions under conditions of regulated filling. A poor correlation was seen between lymph pressure fluctuations and lymphangion contractions, as well as between the rate of lymphangion filling and contractions. We observed stable spontaneous contractions of both bovine and rat mesenteric lymphangions at 0 cm of water intralymphatic pressure, and in the absence of distension stimuli (both radial and axial stretch). Under conditions of low filling, contractions of bovine lymphangions produced negative intralymphatic pressure and a suction effect. The automatism is an inherent feature of the lymphatic smooth muscle cells. Distension of the lymphangion modulates the electrical characteristics of the membranes and thus the contractile mechanisms in lymphangions.

Role of phospholipase C, protein kinase C, and calcium in VEGF-induced venular hyperpermeability.

We previously demonstrated that vascular endothelial growth factor (VEGF)-elicited increase in the permeability of coronary venules was blocked by the nitric oxide (NO) synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). The aim of this study was to delineate in more detail the signaling pathways upstream from NO production in VEGF-induced venular hyperpermeability. The apparent permeability coefficient of albumin (Pa) and endothelial cytosolic Ca2+ concentration ([Ca2+]i) were measured in intact perfused porcine coronary venules using fluorescence microscopy. VEGF (10(-10) M) induced a two- to threefold increase in Pa, which was blocked by a monoclonal antibody directed against the VEGF receptor Flk-1/KDR, the phospholipase C (PLC) antagonist U-73122, or the protein kinase C (PKC) antagonist bisindolylmaleimide (BIM). In 12 venules that displayed the [Ca2+]i response to bradykinin (10(-6) M) and ionomycin (10(-6) M), only 4 vessels responded to VEGF with a transient increase in [Ca2+]i. Furthermore, Western blot analysis of cultured human umbilical vein endothelial cells showed that VEGF increased tyrosine phosphorylation of PLC-gamma and serine phosphorylation of endothelial constitutive NO synthase (ecNOS). The hyperphosphorylation of PLC-gamma was greatly attenuated by the KDR receptor antibody and U-73122, but not by BIM or L-NMMA. In contrast, U-73122 and BIM were able to inhibit VEGF-elicited serine phosphorylation of ecNOS. The results suggest that VEGF induces venular hyperpermeability through a KDR receptor-mediated activation of PLC. In turn, ecNOS is activated by PLC-mediated PKC and/or cytosolic Ca2+ elevation stimulation.

Effect of the non-peptide blocker (+/-) CP 96,345 on the cellular mechanism involved in the response to NK1 receptor stimulation in human skin fibroblasts.

The effect of the selective non-peptide antagonist for NK1 receptors (+/-)CP 96,345 on cellular transduction mechanisms elicited by the NK1 selective agonist [Sar9]-substance P-sulfone ([Sar9]-SP) was investigated in a stabilized culture of human skin fibroblasts (HF) and compared to the effects of two peptide antagonists, FK 888 and GR 82, 334. The exposure of the cells to [Sar9]-SP (100 nM) produced an early increase in inositol 1,4,5-trisphosphate (IP3) level, which peaked after 6 s, and a later rise in cellular inositol 1-phosphate (IP1) content which reached the maximum level in 15 min. The cAMP level was not significantly modified. The increase in IP1 was greatly reduced, at approximately the same extent by the 10 min pretreatment with a concentration of (+/-)CP 96,345 (100 nM) 10 times smaller than that of FK 888 and GR 82,334 (1 microM). The cytosolic Ca2+ mobilization in response to the NK1 agonist was monitored both by spectrofluorimetric and single-cell image analysis determinations on adherent cells loaded with the Ca(2+)-sensitive fluorescent indicators Fura-2/AM and Indo-1, respectively. [Sar9]-SP (100 nM) produced a rapid increase in the intracellular Ca2+ level in Fura-2/AM loaded cells. Cytosolic Ca2+ mobilization, measured by single-cell image analysis, indicated a concentration-dependent increase in both the ratio and in the number of cells responding to [Sar9]-SP. Either the non-peptide or the peptide selective NK1 receptor antagonists inhibited the increase in Ca2+ level in both the assays. In the spectrofluorimetric experiments the antagonizing effects of (+/-)CP 96,345 (1-100 nM), FK 888 (10 nM-1 microM) and GR 82,334 (10 nM-1 microM) were concentration-dependent. Moreover, the non-peptide antagonist was more potent than the two peptide antagonists, producing an 82.5% inhibition of Ca2+ mobilization at a concentration (10 nM) at which FK 888 and GR 82,334 decreased the response by only 62.3 and 60%, respectively. Stimulation of phosphatidylinositol turnover and calcium mobilization were also induced by 10 nM bradykinin; these effects were influenced neither by the previous administration of the NK1 receptor agonist nor by the three antagonists tested. These results demonstrate that the cellular transduction mechanisms induced in human skin fibroblasts by NK1 receptor stimulation are specifically and effectively antagonized by (+/-)CP 96,345, and that this non-peptide antagonist is more potent than the two peptide antagonists tested.

Lymphatic microcirculation.

OBJECTIVE: The importance of the lymphatic system during inflammatory response is underscored by the discovery that numerous inflammatory mediators alter the lymph pump. Inhibition of the lymph pump will reduce the lymphatic outflow from the interstitial space and thus initiate the lymphatic generation of edema. We evaluated the effect that reactive oxygen metabolites have on the contractile activity of lymphatic vessels. METHODS: Reactive oxygen metabolites are produced during inflammation and have been implicated in a number of pathologies. Exposure to reactive oxygen metabolites inhibited the lymph pump flow in a time- and concentration-dependent fashion by decreasing contraction frequency, strength, and propagation. RESULTS: Substance P (SP) (1.0 microM) produced the following changes (% control): reductions in diastolic diameter (56%), systolic diameter (47%), and stroke volume of the lymph pump (62%); a large increase in lymphatic contraction frequency (640%); and a modest increase in lymph pump flow (43%). SP also stimulated quiescent vessels to develop typical contraction-relaxation patterns. These effects may serve to minimize the formation of edema in the face of inflammatory edemagenic conditions that are produced by SP. CONCLUSIONS: We concluded that reactive oxygen metabolites significantly inhibit the active lymph pump and that this inhibition could be an important contributing factor in the formation of interstitial edema during inflammation We have also determined the effects of a putative mediator of inflammation (SP) on the lymphatic pumping function.

Interaction of neutrophils and endothelium in isolated coronary venules and arterioles.

This study reports measurements of porcine neutrophil dynamics in isolated microvessels. Porcine coronary venules and arterioles were isolated, cannulated, and perfused with fluorescently labeled neutrophils at a series of flow velocities. In venules (62.50 +/- 5.41 microns diam) under control conditions, rolling neutrophils were often observed at intraluminal flow velocities ranging from 600 to 6,000 microns/s, and the rolling fraction varied inversely as a function of flow velocity. There was no significant adherence under the control conditions at any of the various flow velocities. Pretreatment of the neutrophils with human recombinant complement 5a (C5a, 10(-8) M) increased adherence at low flow velocities but did not alter the rolling fraction. In contrast to venules, rolling neutrophils were not observed in arterioles (58.80 +/- 5.6 microns diam). Furthermore, neutrophils that were pretreated with C5a did not adhere to the arteriolar endothelium even at low flow velocities. We suggest that 1) isolated microvessels perfused with fluorescently labeled neutrophils are suitable models for the study of the interaction between neutrophils and the microvascular endothelium, 2) shear force plays an important role in neutrophil rolling in coronary venules but is not the major factor that prevents neutrophil rolling and adherence in arterioles, and 3) C5a causes neutrophil adherence in venules but not in arterioles, indicating that different mechanisms underlie the interaction between neutrophils and endothelium in venules and arterioles.

Inhibition of the active lymph pump in rat mesenteric lymphatics by hydrogen peroxide.

The lymphatic system plays an important role in the regulation of fluid and macromolecular exchange. It is a key "safety factor" against the formation of gross edema. Spontaneous contractions in collecting lymphatics of the rat intestine are necessary for the normal transportation of lymph. Hydrogen peroxide is one of the compounds released in inflammation. Therefore, the effects of H2O2 on the pumping activity of spontaneously contracting lymphatics were evaluated in the anesthetized rat (n = 16). Diameter oscillations of the mesenteric collecting lymphatics were monitored before and after the application of H2O2 (4 and 37 microM). The activity of the lymph pump was evaluated using: contraction frequency (F), stroke volume (SV), ejection fraction (EF), and lymph pump flow (LPF). These parameters were determined from the lymphatic diameter tracings. The following changes in lymphatic activity were seen after a 20 minute exposure to 37 microM H2O2: 1) F declined 85%, from 11.6 +/- 1.5 to 1.9 +/- 1.9 cpm. 2) SV fell over 93%. 3) EF decreased 93%, from 0.57 +/- .07 to 0.05 +/- .04. 4) LPF fell dramatically (> 95%) from 41.5 +/- 10.5 to 2.6 +/- 2.5 nl/min. In conclusion, H2O2 produced an intense inhibition of the active lymph pump and it is possible that the inhibition of the active pump contributes to the edema which occurs during inflammation.

Calcium entry, mobilization, and extrusion in postcapillary venular endothelium exposed to bradykinin.

The effect of bradykinin (BK) on cytosolic calcium in coronary venular endothelial cells (CVEC) was studied using the intracellular calcium indicator indo 1. At normal extracellular calcium levels, CVEC responded to BK at concentrations as low as 0.1 pM; maximum cytosolic calcium spikes occurred at 10 nM. In calcium-free medium, poststimulation cytosolic calcium concentration returned to levels below prestimulation values, implying that BK modulates calcium extrusion mechanisms that are normally masked by calcium influx into the cell. To test this hypothesis, we depleted internal stores of calcium using two approaches: preconditioning or blockade of the endoplasmic reticulum calcium pump with the sesquiterpene lactone, thapsigargin. Depletion by preconditioning consisted of two prior doses of BK followed by a third stimulus of the agonist. Under these conditions, the final dose of BK caused a fall, rather than rise, in cytosolic calcium. Thapsigargin blocked the endoplasmic reticulum calcium pump, leading to a steady-state rise in intracellular calcium concentration. Subsequent exposure of these cells to BK also led to a fall in cytosolic calcium. The preconditioning and thapsigargin studies are consistent with a modulation of calcium extrusion processes by BK in CVEC. The signals responsible for this modulation are unknown.

Permeability to albumin in isolated coronary venules.

This study reports measurements of albumin permeability in isolated coronary venules. The isolated microvessel technique allows the quantification of transmural exchange of macromolecules under tightly controlled physical and chemical conditions. Transvenular exchange of albumin was studied in isolated coronary venules during alterations in filtration rate caused by changes in intravascular pressure. The apparent permeability coefficient of albumin (Pa) at an intraluminal pressure of 11 cmH2O was 3.92 +/- 0.43 x 10(-6) cm/s. Elevating intraluminal pressure to 16 and 21 cmH2O increased Pa to 5.13 +/- 0.57 x 10(-6) and 6.78 +/- 0.66 x 10(-6) cm/s, respectively. Calculation of the true diffusive permeability coefficient of albumin (Pd) at zero filtration rate was 1.54 x 10(-6) cm/s. The product of hydraulic conductance (Lp) and (1 - sigma), where sigma is the solute reflection coefficient, was 3.25 x 10(-7) cm.s-1 x cmH2O-1. At a net filtration pressure of 4-5 cmH2O, diffusion accounts for > 60% of total albumin transport across the venular wall. Transmural albumin flux is very sensitive to filtration rate, rising 6.7% for each cmH2O elevation of net filtration pressure. At 11 cmH2O net filtration pressure, convection accounts for nearly 70% of net albumin extravasation from the venular lumen. We suggest that the isolated coronary venule is a suitable preparation for the study of solute exchange in the heart.

Histamine increases venular permeability via a phospholipase C-NO synthase-guanylate cyclase cascade.

In this study, we hypothesized that histaminergic increases in venular permeability result from a cascade triggered by activation of phospholipase C (PLC), inducing the synthesis of nitric oxide (NO) and activating guanylate cyclase. The apparent permeability coefficient to albumin (Pa) was measured in isolated porcine coronary venules subjected to constant flow and hydrostatic and oncotic pressures. Histamine (2.5, 5, and 10 microM) transiently and progressively increased Pa. The PLC inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC; 100 microM) decreased baseline permeability and abolished the effect of histamine. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 10 microM) and the guanylate cyclase inhibitor 6-anilinoquinoline-5,8-quinone (LY 83583; 10 microM) also blocked the histamine-induced hyperpermeability. L-Arginine (3 mM) reversed the inhibition by L-NMMA. NG-monomethyl-D-arginine did not influence the effect of histamine. Furthermore, sodium nitroprusside (10 microM) augmented Pa by two- to threefold; this effect was blocked in the presence of LY 83583 but not altered in the presence of NCDC. The results suggest that histamine increases coronary venular permeability by a direct action on the venular endothelial cells through a PLC-NO synthase-guanylate cyclase-signaling cascade.

Distribution, propagation, and coordination of contractile activity in lymphatics.

The propagation and coordination of lymphatic contractions were studied in the mesentery of the rat small intestine using in situ microscopic observation. Indexes of lymphatic diameter were simultaneously measured at two adjacent lymphangions in spontaneously contracting lymphatics (n = 51). Diameter index, contraction frequency, and the percentage of the intersegmental contractions that were propagated and coordinated (PP) were determined at both sites. The conduction velocity of the contractile activity and the percentage of the coordinated contractions that were propagated both antegrade to the direction of lymph flow and retrograde to the flow stream were determined. The results indicate that 1) 80-90% of the lymphatic contractions in the vessels we evaluated were propagated, 2) the wave of contractile activity propagated both centrally and peripherally, and 3) the conduction velocity of the contractile activity was approximately 4-8 mm/s. We tested the hypothesis that gap junctional communication is responsible for the coordination of the contractile event. To accomplish this, we used the gap junction blockers n-heptanol and oleic acid. PP was 90 +/- 4% under normal conditions and fell to a minimum value of 55 +/- 7% during the gap junction blockade. These results indicate that gap junctional communication played an important role in the propagation and coordination of contractions that occurred in spontaneously active lymphatics.