Biomedical subjects

H B Hechtman

Publications and source records attributed to H B Hechtman.

At least 91 records · Page 5Linked to original sources

PubMed1990

Interleukin-2-induced lung permeability is mediated by leukotriene B4.

Interleukin (IL)-2 therapy leads to respiratory dysfunction due to increased vascular permeability. This study examines the role of the chemoattractant, immunomodulator, and permeability-promoting agent leukotriene (LT) B4 in this setting. Sheep with chronic lung lymph fistulae were given IL-2, 10(5) U/kg as an IV bolus (n = 6). Within 2 hours this led to a significant increase in LTB4 levels in both plasma and lung lymph. The mean pulmonary artery pressure (MPAP) rose while the pulmonary artery wedge pressure was unchanged. Arterial oxygen tension (PaO2) fell. Lung lymph flow (QL) was tripled (P less than 0.05) at 3 hours, coinciding with an increase in the lymph/plasma (L/P) protein ratio (P less than 0.05) resulting in an increase in the lymph protein clearance (P less than 0.05), data documenting increased microvascular permeability to protein. Mild leukopenia and thrombocytopenia (P less than 0.05) occurred. Body temperature rose and shaking chills were common. Pretreatment with the lipoxygenase inhibitor diethylcarbamazine (DEC; n = 6) reduced baseline plasma LTB4 levels and prevented the IL-2-induced increases in LTB4 in plasma and lung lymph (P less than 0.05). In contrast to IL-2 treatment alone, DEC blunted the increase in MPAP and prevented the rises in QL (P less than 0.05), L/P protein ratio (P less than 0.05), and lymph protein clearance (P less than 0.05). DEC also prevented the IL-2-induced leukopenia, the fall in platelet count, and the rise in body temperature (P less than 0.05, respectively). Infusion of IL-2 excipient control (n = 5) did not affect plasma or lymph LTB4 levels but there were mild increases in MPAP (P less than 0.05). The QL also rose but this occurred while the L/P protein ratio fell (P less than 0.05). Body temperature rose moderately. The PaO2, leukocyte, and platelet counts were unaffected. These data indicate that IL-2 administration leads to pulmonary dysfunction manifest by pulmonary hypertension and increased vascular permeability, events associated with LTB4 synthesis and prevented by DEC. Leukotriene B4 appears therefore to mediate the IL-2-induced lung injury.

Animals↗

PubMed1990

Neutrophil adherence receptors (CD 18) in ischemia. Dissociation between quantitative cell surface expression and diapedesis mediated by leukotriene B4.

Neutrophils and eicosanoid chemoattractants are centrally involved with ischemia-reperfusion (I/R) injury. The CD 18 complex of adhesive glycoproteins, readily up-regulated by chemoattractants in vitro, is required for polymorphonuclear leukocyte (PMN) adherence to endothelium. This study tests whether CD 18 is up-regulated by ischemia in vivo and its role in mediating PMN diapedesis. Anesthetized rabbits underwent 3 h of bilateral hindlimb tourniquet ischemia (n = 16). Ten min after tourniquet release, levels of plasma leukotriene (LT)B4 increased to 390 +/- 62 pg/ml (mean +/- SE), higher than 134 +/- 26 pg/ml in control rabbits (n = 13, p less than 0.01). Aliquots of plasma were added to whole blood from normal rabbits (n = 6) for flow cytometric analysis of neutrophils with the CD 18 mAb R 15.7. Addition of I/R plasma failed to demonstrate an increase in surface expression of CD 18. Similarly, no CD 18 up-regulation was observed in vivo upon reperfusion in ischemic animals pretreated with mAb R 15.7 (n = 3). However, I/R plasma when introduced into plastic chambers taped atop dermabrasion sites in normal rabbits (n = 12) resulted in diapedesis, measured by the accumulation after 3 h of 1130 +/- 125 PMN/mm3 in the chambers relative to 120 +/- 31 PMN/mm3 with control plasma (p less than 0.01). Diapedesis in response to I/R plasma was abolished by pretreatment with mAb R 15.7 (less than 5 PMN/mm3, n = 6), was reduced by U 75,302, an LTB4 receptor antagonist (253 +/- 101 PMN/mm3, n = 6) (both p less than 0.01) and was not protein synthesis dependent. These results demonstrate that PMN diapedesis in response to I/R plasma is exclusively dependent upon the CD 18 glycoprotein complex by an LTB4-dependent mechanism, despite the fact that CD 18 is not up-regulated on circulating PMN in ischemia. These data indirectly indicate the functional importance of conformational changes of CD 18 in determining PMN adhesion.

Animals↗

PubMed1990

Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells.

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.

Animals↗

PubMed1990

Inflammatory agonists that increase microvascular permeability in vivo stimulate cultured pulmonary microvessel endothelial cell contraction.

Bovine pulmonary microvessel endothelial cells grown on a flexible substrate contract upon the addition of angiotensin II, thrombin, bradykinin, and U44069, a stable analogue of thromboxane A2. All these agents promote inflammation and increase paracellular permeability in vivo or in vitro. The contractile response is mediated by intracellular and extracellular free calcium: the response is inhibited by TMB-8, an intracellular Ca2+ chelator, and EGTA. Contraction is inhibited by trifluoroperazine, a Ca2(+)-calmodulin antagonist, and by ML-7, an inhibitor of myosin light-chain kinase. Preincubation with PMA, a protein kinase C activator, prevents contraction by angiotensin II. The inactive analogue 4-alpha-phorbol 12,13-didecanoate does not inhibit contraction. In contrast cAMP, carbacyclin (a stable PGI2 analogue), and isoproterenol, agonists known to stabilize the microvascular barrier against inflammatory agents, relax pulmonary microvessel EC. This direct evidence of the contractile potential of microvessel endothelial cells lends support to the theory that endothelial contraction leads to increased junctional permeability.

Angiotensin II↗

PubMed1990

Modulation of cultured pulmonary microvessel and arterial endothelial cell barrier structure and function by serotonin.

Pulmonary microvessel endothelial cell and pulmonary artery endothelial cell monolayers in tissue culture were treated with serotonin (5-hydroxytryptamine; 5-HT) alone or in conjunction with histamine, bradykinin, the thromboxane analog U-46619, and the actin modulating agent cytochalasin B. After agent treatment, cross-sections through endothelial cell (EC) monolayers were examined by light microscopy and the percentage and widths of intercellular openings were quantitated. To correlate structural changes in the endothelial barrier with an alteration in permeability, EC monolayers cultured on micropore filters were assayed for transit of Evan's blue albumin (EBA) following treatment with vasoactive mediators. 5-HT was found to decrease the patency of endothelial junctions by up to 94%, compared to untreated monolayers, and to prevent or reverse the appearance of interendothelial gaps induced by histamine, bradykinin, U-46619, and cytochalasin B. The 5-HT effect was dose and time dependent, with a maximal increase in junctional apposition observed at a concentration of 10(-6) M for 30 min. This response was significantly blocked by the 5-HT antagonists LSD and ketanserin. The formation or reduction of interendothelial gaps by histamine, bradykinin, and U-46619 and by 5-HT, respectively, was positively correlated to changes in monolayer permeability to EBA. These results suggest that pulmonary edema caused by inflammatory mediators in part may be a consequence of transient increases in pulmonary EC junctional gaps, and that 5-HT may contribute to the homeostatic maintenance of endothelial barrier integrity.

Animals↗

PubMed1990

Ischemia activates neutrophils but inhibits their local and remote diapedesis.

Hindlimb ischemia and reperfusion results in local limb and distant lung injury. This study tests whether the mechanism of injury is by ischemia mediated polymorphonuclear leukocyte (PMN) activation and diapedesis. Anesthetized rabbits were subjected to three hours of hindlimb ischemia (n = 8) or sham ischemia (n = 4). PMN derived solely from the reperfused ischemic limb and assayed flow cytometrically displayed an oxidative burst of 135 /- 8 fentamoles dichlorofluorescein (fmDCF)/cell compared to preischemc levels of 74 +/- 14 fmDCF/cell (p less than 0.05). Additional aliquots of isolated neutrophils were treated with phorbol myristate acetate (PMA) 10(-7) M. In contrast to a 162% increase in oxidative burst before ischemia, neutrophils at ten minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Plasma collected from the ischemic hindlimb at ten minutes of reperfusion when introduced into an abraded skin chamber or intratracheally induced diapedesis in nonischemic animals. PMN accumulations in the skin chamber were 1636 +/- 258 PMN/mm3 after three hours (n = 8) compared to 63 +/- 18 PMN/mm3 induced by sham plasma (n = 4, p less than 0.05). Introduction of ischemic plasma intratracheally into a lobar bronchus (n = 4) induced PMN accumulations after three hours, measured by bronchoalveolar lavage fluid of 19 +/- 2 X 10(4) PMN/mm3 compared to 5 +/- 1 X 10(4) PMN/mm3 with sham plasma (n = 4, p less than 0.05). Diapedesis was completely prevented (0-3 PMN/mm3, p less than 0.05) by introducing ischemic plasma into skin chambers in animals whose hindlimbs had been made ischemic (n = 6) or into chambers located on skin regions that had been previously made ischemic (n = 6). Similarly after hindlimb ischemia, lavage of the lung with ischemic plasma yielded few PMN 0-3/mm3 (p less than 0.05). These data indicate that ischemia and reperfusion lead to generation of a circulating component in plasma that causes an oxidative burst in PMN and inhibits their diapedesis but promotes diapedesis when applied extravascularly to a naive animal.

Animals↗

PubMed1990

Tumor necrosis factor-alpha mediates acid aspiration-induced systemic organ injury.

Acid aspiration-induced systemic organ injury is mediated by the sequestration of activated neutrophils (PMN). In other settings cytokines have been shown to increase neutrophil-endothelial adhesion, a requisite for injury. This study tests whether the systemic leukosequestration and permeability following localized aspiration is mediated by tumor necrosis factor (TNF)-alpha-induced synthesis of an adhesion protein. Anesthetized rats underwent tracheostomy and insertion of a fine-bore cannula into the anterior segment of the left lung. This was followed by the instillation of either 0.1 mL 0.1 N HCI (n = 18) or 0.1 mL saline in control rats (n = 18). Localized aspiration induced generalized pulmonary leukosequestration with 95 PMN/10 high-power fields (HPF) in the aspirated lung and 46 PMN/10 HPF in the nonaspirated lung, higher than control values of 7 PMN/10 HPF and 5 PMN/10 HPF in saline- and nonsaline-aspirated sides, respectively (p less than 0.05). The leukosequestration was associated with permeability edema shown by increased protein concentrations in bronchoalveolar lavage (BAL) of 3900 micrograms/mL in the aspirated and 2680 micrograms/mL in the nonaspirated side, higher than saline with 482 micrograms/mL and 411 micrograms/mL, respectively (p less than 0.05). There was generalized pulmonary edema following aspiration measured by increase in wet-to-dry weight ratios (w/d) of 6.6 in the aspirated and 5.1 in the nonaspirated lung, higher than control values of 3.5 and 3.4, respectively (p less than 0.05). Localized aspiration led to systemic leukosequestration documented by increases in myeloperoxidase activity (units/g tissue) of 2.2 and 1.7 in heart and kidney, higher than control values of 0.3 and 0.4, respectively (p less than 0.05). This event was associated with edema of these organs with w/d ratios of 4.6 and 4.3, relative to control values of 3.0 and 3.4 (p less than 0.05). Treatment of animals (n = 18) 20 minutes after aspiration with anti-TNF-alpha antiserum (rabbit anti-murine) but not normal rabbit serum (n = 18) reduced lung leukosequestration in the aspirated and nonaspirated segments (61 and 32 PMN/10HPF), BAL protein concentration (1490 and 840 micrograms/mL), and w/d ratio (4.3 and 3.7) (all p less than 0.05). In the heart and kidney there were reductions in myeloperoxidase activity (0.7 and 0.6) and w/d ratio (3.5 and 3.6) (both p less than 0.05). Treatment of rabbits (n = 18) with the protein synthesis inhibitor cycloheximide, 0.2 mg/kg/hr was as effective as TNF-alpha antiserum in modifying aspiration injury.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Involvement of thromboxane and neutrophils in multiple-system organ edema with interleukin-2.

Interleukin-2 (IL-2) produces toxicity characterized by generalized edema within 24 hours. This study tests whether the rate of IL-2 administration modulates the onset of edema and examines thromboxane (Tx) and neutrophils as possible mediators of this event. Recombinant human IL-2, 10(5) U (n = 7), 10(6) U (n = 9), or vehicle (n = 8) were given to anesthetized rats intravenously during a period of 1 hour. At 6 hours edema, as measured by increase in wet to dry weight (w/d) ratio, was present in the heart, liver, and kidney, with 10(5) U IL-2 and in the lung, heart, liver and kidney, with 10(6) U IL-2, relative to values with vehicle-infused controls (all p less than 0.05). With a 1-hour infusion of 10(6) U IL-2, there was an increase in plasma thromboxane (Tx)B2 level to 1290 +/- 245 pg/mL, higher than 481 +/- 93 pg/mL in control rats (p less than 0.05); lung polymorphonuclear leukocyte (PMN) sequestration of 53 +/- 7 PMN/10 higher-power fields (HPF) relative to 23 +/- 2 PMN/10 HPF in controls (p less than 0.05); and increased bronchoalveolar lavage (BAL) fluid protein concentration of 1970 +/- 210 micrograms/mL relative to 460 micrograms/mL in controls (p less than 0.05). When 10(6) U IL-2 was given as a 1-minute intravenous bolus (n = 9), edema was not demonstrated, plasma TxB2 levels were similar to controls, there was no leukosequestration, and BAL protein levels were normal. These data indicate that a constant infusion but not the rapid bolus administration of IL-2 produces in rats multiple-system organ edema, increased plasma TxB2, sequestration of PMNs, and microvascular permeability. These findings may explain the early toxicity seen in patients given high-dose IL-2 in cancer treatment.

6-Ketoprostaglandin F1 alpha↗

PubMed1990

Attenuation of acid aspiration edema with phalloidin.

Acid aspiration leads to pulmonary endothelial and epithelial cell (EC/EpC) injury characterized by increased permeability and polymorphonuclear (PMN) leukocyte diapedesis. Actin in the EC/EpC cytoskeleton has been shown to play a significant role in maintenance of the microvascular junction barrier. This study tests indirectly whether the development of permeability and diapedesis following acid aspiration is via disruption of the pulmonary cytoskeleton. Manipulation was achieved by the actin microfilament assembler phalloidin. Anesthetized rats (n = 88) underwent segmental lung installation of 0.1 ml saline or phalloidin (2 x 10(-6) M). Twenty minutes later 0.1 N HCl, saline, or phalloidin was introduced. After 3 h there was an increase in wet-to-dry weight (W/D) ratio of 6.6 and 5.1 in the HCl-injected and noninjected sides, protein concentration, 3,970 and 2,530 micrograms/ml, and accumulation of 93 PMN/ml (x 10(4] in bronchoalveolar lavage (BAL) of the HCl-injected lung. These values were higher than control animals. Local pretreatment with phalloidin attenuated acid-induced localized but not generalized permeability with reduction in W/D ratio, BAL protein concentration, and diapedesis (P less than 0.05). Acid injection into airways also led to elevated thromboxane B2 and leukotriene B4 levels in plasma and BAL (P less than 0.05) and generalized lung leukosequestration, events not affected by phalloidin. Taken together, these data suggest that acid aspiration lung injury is determined largely by loss of integrity of the pulmonary EC/EpC cytoskeleton with resultant loss of barrier function.

Administration, Inhalation↗

PubMed1990

Ischemia-induced neutrophil activation and diapedesis is lipoxygenase dependent.

Ischemia and reperfusion lead to eicosanoid- and neutrophil (PMN)-dependent injury. This study tests the role of ischemia-induced lipoxygenase activity in mediating PMN activation and diapedesis. Anesthetized rabbits (n = 8) underwent 3 hours of bilateral hindlimb ischemia. At 10 minutes of reperfusion, leukotriene B4 (LTB4) levels in femoral venous effluent were 0.49 +/- 0.05 ng/ml compared with 0.04 +/- 0.07 ng/ml in sham-treated animals (n = 10) (p less than 0.05). Intracellular H2O2 production of circulating PMNs assayed flow cytometrically by dichlorofluorescein (DCF) oxidation, increased from a preischemic value of 74 +/- 14 femtomoles DCF/cell to 135 +/- 8 fmol DCF/cell (p less than 0.05). PMNs were treated with phorbol myristate acetate (PMA), 10(-7) mol/L. In contrast to a 162% increase in H2O2 production before ischemia, PMNs at 10 minutes of reperfusion had an enhanced response to PMA of 336% (p less than 0.05). Addition of authentic LTB4 (0.5 ng/ml) to PMN from sham-treated animals led to their activation, manifest by an oxidative burst, 127 +/- 12 fmol DCF/cell, and an enhanced response of 337% to PMA stimulation. To study diapedesis, plasma collected at 10 minutes of reperfusion was introduced into plastic chambers taped atop skin abrasions in rabbits (n = 8). After 3 hours, 1610 +/- 246 PMN/mm3 accumulated and LTB4 levels in blister fluid were 0.83 +/- 0.03 ng/ml, higher than values of 44 +/- 23 PMN/mm3 (p less than 0.05) and 0.04 +/- 0.03 ng LTB4/ml (p less than 0.05) with saline solution and 68 +/- 16 PMN/mm3 (p less than 0.05) and 0.19 +/- 0.02 ng/ml (p less than 0.05) with nonischemic plasma. The introduction of LTB4, 3.3 ng/ml, into the chambers resulted in an accumulation of 536 +/- 352 PMN/mm3 (p less than 0.05). Pretreatment of animals before hindlimb ischemia (n = 5) with the lipoxygenase inhibitor diethylcarbamazine abolished PMN activation (51 +/- 12 fmol DCF/cell) and ischemic plasma-induced diapedesis into the plastic chamber (38 +/- 18 PMN/mm3). Pretreatment of nonischemic animals (n = 13) used for the dermabrasion bioassay with diethylcarbamazine abolished diapedesis into the plastic chambers induced by ischemic plasma (n = 5) (32 +/- 24 PMN/mm3) or LTB4 (n = 3) (36 +/- 28 PMN/mm3). These data indicate that PMN activation after reperfusion of ischemic tissue is mediated by a lipoxygenase product, perhaps LTB4, and that both reperfusion plasma and authentic LTB4 induce diapedesis by stimulating de novo lipoxygenase activity.

Animals↗

PubMed1989

Role of thromboxane in interleukin 2-induced lung injury in sheep.

Interleukin (IL)-2 administration leads to respiratory dysfunction due to increased vascular permeability. This study examines the role of thromboxane (Tx)A2 in IL-2 induced lung injury in sheep with chronic lung lymph fistulae. This preparation enables evaluation of permeability prior to the development of gross edema. IL-2, 10(5) units/kg (n = 6), or its excipient control (n = 5) was given as an i.v. bolus over 2 min. After 2 h of IL-2 administration, plasma TxB2 increased from 168 to 388 pg/ml (P less than 0.05) and lung lymph TxB2 from 235 to 694 pg/ml (P less than 0.05). Mean pulmonary artery pressure (MPAP) rose from 13 to 29 mm of Hg (P less than 0.05) at 30 min and remained elevated for 4 h while the pulmonary artery wedge pressure was unchanged at 4 mm of Hg. Arterial oxygen tension (PaO2) fell from 88 to 77 mm of Hg (P less than 0.05). Lung lymph flow (QL) rose from 2.2 to 3.8 ml/30 min (P less than 0.05) at 1 h and to 6.4 ml/30 min at 3 h. This rise coincided with an increase in the lymph/plasma (L/P) protein ratio from 0.67 to 0.77 (P less than 0.05). In contrast, the non-IL-2-infused sheep (n = 3) recruitment of the lung vasculature by left atrial balloon inflation led to a rise in QL from 2.4 to 8.2 ml/30 min, whereas the L/P ratio declined from 0.62 to 0.25, suggesting that the protein-rich lymph flow after IL-2 administration reflected increased microvascular permeability. In further proof of an increase in permeability, IL-2 administration into sheep (n = 2) with an inflated left atrial balloon led, after a pressure-independent L/P protein ratio had been achieved, to an increase in L/P protein ratio and decrease in protein reflection coefficient. At 2 h after IL-2, the blood leukocyte count fell from 8156 to 4375/mm3 (P less than 0.05) primarily due to a 73% drop in lymphocytes. The platelet count declined from 292 to 184 x 10(3)/mm3 (P less than 0.05). Body temperature rose from 38.9-40.3 degrees C (P less than 0.05), and shaking chills were common. Pretreatment with the Tx synthetase inhibitor OKY 046 (n = 7) lowered baseline plasma and lymph TxB2 levels to 22 and 52 pg/ml (P less than 0.05) and prevented the IL-2-induced increase in plasma and lung lymph TxB2 (P less than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Reperfusion pulmonary edema.

Reperfusion following lower-torso ischemia in humans leads to respiratory failure manifest by pulmonary hypertension, hypoxemia, and noncardiogenic pulmonary edema. The mechanism of injury has been studied in the sheep lung lymph preparation, where it has been demonstrated that the reperfusion resulting in pulmonary edema is due to an increase in microvascular permeability of the lung to protein. This respiratory failure caused by reperfusion appears to be an inflammatory reaction associated with intravascular release of the chemoattractants leukotriene B4 and thromboxane. Histological studies of the lung in experimental animals revealed significant accumulation of neutrophils but not platelets in alveolar capillaries. We conclude that thromboxane generated and released from the ischemic tissue is responsible for the transient pulmonary hypertension. Second, it is likely that the chemoattractants are responsible for leukosequestration, and, third, neutrophils, oxygen-derived free radicals, and thromboxane moderate the altered lung permeability.

Animals↗

PubMed1989

Pulmonary microvascular endothelial cell contractility on silicone rubber substrate.

Endothelial cell (EC) motility may contribute to the regulation of microvascular perfusion and/or paracellular permeability. The experiments reported herein demonstrate that bovine pulmonary microvessel EC can reversibly deform a silicone substrate in response to agents known to contract and relax smooth muscle cells. Contracting pulmonary microvessel EC exerted a tension that created wrinkles in the underlying deformable substrate. Relaxation and loss of tension were revealed by the disappearance of these wrinkles without loss of cell adhesion to the substratum. Angiotensin II (Ang II) and bradykinin stimulated pulmonary microvessel EC to contract within 3 to 8 min in a Ca2+-dependent fashion. The peak of contraction at 10 to 20 min was followed by relaxation. Forskolin and sodium nitroprusside (SNP) initiated relaxation of the microvessel EC within 3 to 10 min respectively. Relaxed EC contracted following the addition of Ang II, also within 3 min. Dibutyryl cAMP, dibutyryl cGMP, and the photoactivated internalized "caged" cAMP and cGMP promoted EC relaxation in a manner similar to forskolin and SNP. Increases in the intracellular concentration of inositol triphosphate (IP3) with the photoactivated IP3 complex promoted EC contraction in 2 min with a peak at 7 min. The contraction was followed by relaxation, which occurred at 20-25 min. Neither bovine pulmonary artery nor retinal microvessel EC, used as controls, contracted under these experimental conditions. One could speculate that this unique contractile property of pulmonary microvessel EC as observed in vitro may play a regulatory role in vivo, in local perfusion and/or in intercellular gap regulation.

Animals↗

PubMed1989

Modulation of phospholipase A2 lytic activity by actin and myosin.

Prostacyclin (PGI2) production is closely coupled with endothelial cell shape and F-actin distribution in vitro. These findings may implicate cytoskeletal constituents in a mechanism regulating eicosanoid metabolism. To determine the potential for such a regulatory mechanism, cytoskeletal protein effects on the rate-limiting eicosanoid cascade enzyme (phospholipase A2; PLA2) were studied. Membrane phospholipid degradation was indirectly determined by spectrophotometric measurement of PLA2-induced rat red blood cell ghost (RBC-G) hemolysis. PLA2 was incubated with actin (skeletal, smooth, or nonmuscle cell) at a nonmuscle cell concentration (100 microM) and then exposed to the RBC-G. Comparisons in the presence or absence of actin revealed that F-actin stimulated whereas G-actin suppressed PLA2 lytic behavior significantly (P less than 0.05). When a 10: or 100:1 F-actin to myosin ratio was used, the F-actin stimulatory effect was significantly (P less than 0.05) reduced. These findings suggest that the in vitro correlation between PGI2 production and endothelial cell shape may be the result of PLA2 regulation by cytoskeletal elements that impart cellular form.

Actins↗

PubMed1989

The endothelial cell cytoskeleton modulates extravascular polymorphonuclear leukocyte accumulations in vivo.

Actin microfilaments, key elements in the endothelial cell (EC) cytoskeleton, have been noted in in vitro studies to play a modulating role in the diapedesis of polymorphonuclear leukocytes (PMN). The role of the cytoskeleton in PMN diapedesis in vivo was the subject of this study. Small skin abrasions were produced in rabbits. Cytoskeletal manipulation was accomplished by local application of phalloidin which promotes microfilament assembly or cytochalasin B which causes their disassembly, prior to addition of a chemotaxin. When the initial treatment to the abrasion site was saline, the secondary addition of more saline resulted in PMN accumulations, expressed as PMN/mm3 of 36 +/- 19, while 10(-4) M cytochalasin B led to 91 +/- 32 (P less than 0.05). Secondary addition of chemotaxins or histamine also led to significant PMN accumulations of 169 +/- 54 with 10(-8) M leukotriene (LT) B4, 318 +/- 85 with zymosan-activated plasma (ZAP), and 158 +/- 48 with 10(-4) M histamine. When the initial treatment was 10(-8) M phalloidin, PMN accumulations were reduced to 31 +/- 22 with cytochalasin B (P less than 0.05 relative to saline as initial treatment); 63 +/- 43 with LTB4 (P less than 0.05); 137 +/- 48 with ZAP (P less than 0.05); and 51 +/- 35 with histamine (P less than 0.05). In contrast, initial blister treatment with cytochalasin B rather than saline increased PMN accumulations in response to LTB4, 291 +/- 71 (P less than 0.05), and histamine, 270 +/- 56 (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Vasoactive amines and eicosanoids interactively regulate both polymorphonuclear leukocyte diapedesis and albumin permeability in vitro.

Previously we reported that cultured endothelial cells (ECs) can promote or inhibit polymorphonuclear leukocyte (PMN) diapedesis and albumin permeability in vitro by altering monolayer intercellular integrity (an activity influenced by pretreatment with exogenous amines). Endothelial eicosanoid release was also seen to stimulate both PMN motility and diapedesis. We now demonstrate that these endothelial activities are related. Thromboxane (Tx) B2 pretreatment of ECs results in increased diapedesis and permeability across the monolayers whereas 6-keto-PGF1 alpha pretreatment has the opposite effects, demonstrating that these eicosanoids exert direct effects upon ECs, in addition to their direct effects upon PMNs as previously described. Norepinephrine (NE) or serotonin (5HT) pretreatment of ECs inhibits the release of TxB2 and 6-keto-PGF1 alpha, with the result that the stimulation of PMN motility by these EC metabolites is eliminated. In contrast, histamine increases the endothelial release of eicosanoids, resulting in a further increase in PMN motility. We conclude that histamine directly reduces EC monolayer integrity (by altering the endothelial cytoskeleton) and also increases eicosanoid release, actions which both enhance PMN motility and further reduce monolayer integrity. Conversely, NE and 5HT both increase intercellular integrity and decrease eicosanoid release, thereby decreasing PMN motility, diapedesis, and albumin permeability.

6-Ketoprostaglandin F1 alpha↗

PubMed1989

Endothelial cell junctional integrity modulation by serotonin: an ultrastructural analysis.

We have reported previously that exogenous serotonin (5-hydroxytryptamine, 5-HT) alters cultured bovine aortic endothelial cell (BAEC) structural integrity by modulating the assembly of stress fibers. In the present study a 5-HT stimulus-coupled change in BAEC junctional integrity was quantitated by determining the width and percentage of intercellular openings in a monolayer. BAEC treated with 5-HT at concentrations of 10(-9) M to 10(-3) M caused a significant dose-dependent decrease in interendothelial cell junctional openings compared to controls, with the greatest reduction induced at 10(-6) M (92% from control). Treatment of BAEC with histamine (10(-4) M) increased the junctional openings by 82% when compared to controls. This change could be prevented by either pretreatment of the monolayers with 5-HT or by adding 5-HT in conjunction with the histamine. To assess a direct interaction of 5-HT with actin filaments, cultured BAEC monolayers were extracted, treated with 5-HT, and processed for immunocytochemical localization of 5-HT using the Avidin-Biotin method. Electron microscopy revealed 5-HT antibody bound to actin filaments and dense in areas of filament intersection, which implies a role for internalized 5-HT in stimulating the assembly of an actin filament network. Collectively, these results suggest that 5-HT helps to regulate the endothelial junctional barrier by promoting actin filament formation and stability, which may in turn increase the junctional apposition between endothelial cells.

Actins↗

PubMed1989

The rapid induction by interleukin-2 of pulmonary microvascular permeability.

The clinical use of interleukin-2 (IL-2) is limited by severe cardiopulmonary dysfunction. This study examines the mechanism of respiratory failure related to IL-2, using sheep with chronic lung lymph fistulae. Awake animals were infused with an intravenous (I.V.) bolus of IL-2 10(5) U/kg (n = 5) or its excipient (EXC) control (n = 3), every 8 hours for 4 to 5 days. Cardiopulmonary function was monitored daily for at least one 8-hour period. Within 2 hours after each IL-2 administration, mean pulmonary arterial pressure (MPAP) rose. On Day 1, the mean rise was from 13 to 26 mmHg (p less than 0.05), and on Day 5, to 29 mmHg (p less than 0.05). MPAP returned to baseline levels after 2-3 hours. Pulmonary arterial wedge pressure was unchanged from 4 mmHg. There were transient falls in arterial oxygen tension, from 88 to 77 mmHg on Day 1 and to 73 mmHg (p less than 0.05) on Day 5. Lung lymph flow (QL) rose from 2.4 to 6.8 ml/30 minutes (p less than 0.05) on Day 1, and from 4.7 to 10.2 ml/30 minutes (p less than 0.05) on Day 5, whereas the lymph/plasma protein ratio increased on Day 1 from 0.69 to 0.83 (p less than 0.05) and from 0.63 to 0.71 (p less than 0.05) on Day 5. This documents an increase in pulmonary microvascular permeability. Thromboxane (Tx)B2 levels increased transiently after each IL-2 injection in plasma from 195 to 340 pg/ml (p less than 0.05) and in lung lymph from 222 to 772 pg/ml (p less than 0.05) on Day 1, and to similar levels on Day 5. There was a progressive rise in cardiac output from 5.7 to 8.6 1/minute (p less than 0.05) during the 5 days of infusion. Systemic blood pressure did not change. Temperature rose from 39.1 to 41.2 C (p less than 0.05), and shaking chills were common. There was a progressive fall in leukocyte count, from 8.4 to 3.2 X 10(3)/mm3 (p less than 0.05) by Day 5, reflecting a 77% fall in lymphocytes. Lung lymph lymphocyte counts rose, and lymphocyte clearance increased.(ABSTRACT TRUNCATED AT 250 WORDS)

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