Biomedical subjects

F N Miller

Publications and source records attributed to F N Miller.

At least 37 records · Page 2Linked to original sources

PubMed1998

N omega-nitro-L-arginine methyl ester inhibits inflammatory liver injury induced by interleukin-2.

Administration of interleukin-2 (IL-2) for treatment of metastatic disease often results in inflammatory liver injury. Previous studies have implicated increased leukocyte and platelet adhesion and enhanced nitric oxide production as causative factors in the development of IL-2-induced hepatic injury. This study investigated the capacity of N omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthesis inhibitor, to limit IL-2-induced hepatic edema and hepatocellular damage in mice. Using hepatic intravital microscopy, we also examined the effects of L-NAME on IL-2-induced increases in leukocyte and platelet adhesion. Administration of IL-2 increased leukocyte and platelet adhesion in post-sinusoidal venules and decreased hepatic perfusion. Cotreatment with L-NAME had no effect on leukocyte adhesion but increased platelet-endothelial adhesion and microvascular thrombosis. Chronic IL-2 treatment induced hepatic edema and hepatocellular injury. However, coadministration of L-NAME attenuated IL-2-induced edema and completely inhibited hepatocellular damage. These findings suggest that nitric oxide may play a central role in IL-2-induced inflammatory liver injury.

Alanine Transaminase↗

PubMed1998

An increase in endothelial intracellular calcium and F-actin precedes the extravasation of interleukin-2-activated lymphocytes.

OBJECTIVE: Interleukin-2 (IL-2) induces protein leakage from the microcirculation and activates lymphocytes; yet it is unclear how it alters endothelial barrier function. Here, we report of a new continuous monitoring system that allows for the continuous measurement and correlation of endothelial calcium, permeability to albumin, and extravasation of lymphocytes. METHODS: IL-2 activated lymphocytes (IL-2 LYMPH) or unstimulated lymphocytes (LYMPH) were co-incubated with human microvascular endothelial cells (HMVEC). Endothelial albumin permeability, lymphocyte extravasation intracellular calcium mobilization, and f-actin distribution were examined using a new continuous monitoring system. RESULTS: The clearance rate of fluorescein isothiocyanate-labeled-human serum albumin (FITC-HSA) in the presence of IL-2 LYMPH peaked at 20 minutes, whereas the clearance rate of LYMPH peaked at 40 minutes. Approximately 40 minutes after the peak in the clearance rate to albumin, extravasation of carboxyfluorescein-labeled lymphocytes was detected. Peak clearance rates for the extravasation of IL-2 LYMPH occurred at approximately 40 minutes after the addition of the lymphocytes to the HMVEC, whereas the peak clearance rate for the LYMPH occurred at 60 minutes after their addition. Both FITC-HSA and lymphocyte extravasation were measured concurrent to endothelial intracellular calcium mobilization by FURA-2. There was an increase in calcium activation after the addition of IL-2 stimulated lymphocytes (71 +/- 5.1 nmol/L to 185 +/- 18.9 nmol/L) compared with unstimulated lymphocytes (71 +/- 5.1 nmol/L to 110 +/- 12.2 nmol/L). The addition of IL-2 had little or no effect on endothelial actin, whereas the unstimulated lymphocytes and, to a greater extent, IL-2 LYMPH increased the presence of transversing stress fibers and decreased peripheral actin. CONCLUSIONS: The findings reported here suggest that the permeability and extravasation events that occur upon addition of lymphocytes proceeds by a calcium- and actin-dependent mechanism and that incubation of lymphocytes with IL-2 enhances normal lymphocyte mechanisms of extravasation.

Actins↗

PubMed1997

Distinct biological activities of recombinant forms of human interleukin-2 in vivo.

The biological activity of all recombinant forms of interleukin-2 (IL-2) is based upon an in vitro lymphocyte proliferation assay and measured in international units (IU). Numerous in vitro investigations have suggested that there may be different cellular effects of recombinant human IL-2 retaining the natural sequence (nIL-2) as compared to another recombinant form containing a serine substitution at amino acid position 125 ([Ser]IL-2). In the present study we investigated whether nIL-2 and [Ser]IL-2 cause similar patterns of systemic toxicities. C57BL/6 mice were treated with identical doses of either nIL-2 or [Ser]IL-2, as measured in IU, for 3 days and had blood and tissues removed for analysis of lymphocyte activation and organ dysfunction. The administration of nIL-2 had considerably greater effects on lymphocyte activation than did [Ser]IL-2, causing much greater up-regulation of the alpha subunit of the IL-2 receptor and the adhesion molecule lymphocyte function-associated antigen-1. Furthermore, nIL-2 induced more organ edema than did [Ser]IL-2 and caused hepatocellular injury, which was absent in mice treated with [Ser]IL-2. These data demonstrate that equivalent doses, measured in IU, of nIL-2 and [Ser]IL-2 have profoundly different effects on the induction of organ toxicity, suggesting that the IU standard may not be appropriate for the measurement of many in vivo biological activities.

Animals↗

PubMed1997

Arteriolar dilation to endotoxin is increased in copper-deficient rats.

We have previously reported that there is an altered response to mast cell-mediated inflammation in copper-deficient rats. In the current study we determined the microvascular reactivity to inflammatory stimuli with lipopolysacccharide (LPS) during dietary copper restriction. Male Sprague-Dawley rats were fed purified diets which were either copper-adequate (CuA, 6 micrograms Cu/g) or copper-deficient (CuD, 0.4 micrograms Cu/g) for 4 weeks. Rats were anesthetized and the cremaster muscle was prepared for in vivo television microscopy. Arteriolar diameters were measured and then 2.5 mg/kg LPS was injected i.p. In separate groups, animals were pretreated with the NO-synthase inhibitor L-NAME (2 x 10(-4) M), the cyclooxygenase inhibitor ibuprofen (9.6 x 10(-5) M) or the histamine receptor antagonist diphenhydramine (DPH, 10(-6) M). LPS caused arteriolar dilation in both dietary groups with the response being significantly greater in the CuD group. Ibuprofen and DPH but not L-NAME, each significantly reduced but did not block the dilation in the CuD group. Ibuprofen and DPH together blocked the dilation. These results suggest that dietary copper deficiency increases arteriolar dilation to LPS. The mechanism appears to involve a greater response to arachidonic acid metabolites and histamine but not NO.

Animals↗

PubMed1997

Bradykinin antagonizes the effects of alpha-thrombin.

alpha-Thrombin (AT) and bradykinin (BK) are endogenous mediators that are released during an inflammatory response, and could have a synergistic effect on endothelial permeability. Human umbilical vein endothelial cells (HUVEC) were grown on Transwell membranes and then tested for alterations in permeability to fluorescein isothiocyanate-labeled human serum albumin. Addition of 1 microM AT produced a significant increase in the permeability coefficient at 30 minutes from control levels of 1.59 x 10(-6) cm/sec to 4.92 x 10(-6) cm/sec. BK (1 microM) produced a similar increase to 4.46 x 10(-6) cm/sec. For both compounds, permeability remained elevated for 90 minutes. Pre-treatment of the HUVEC with the bradykinin receptor antagonist, Na-adamantaneacetyl-bradykinin (NA-BK) (1 microM), prior to addition of AT, reduced the AT permeability coefficient to 2.69 x 10(-6) cm/sec. Addition of NA-BK (1 microM) for 5 minutes, then BK (1 microM) for 5 minutes, inhibited the effect of BK and of AT (1 microM on permeability, decreasing the permeability coefficient of the endothelial monolayer to control levels (1.62 x 10(-6) cm/sec). AT (1 microM) increased HUVEC intracellular calcium mobilization, as monitored by FURA-2, to 245 nM from control (70 nM), however, pre-treatment with either BK or the bradykinin receptor antagonist decreased the AT induced intracellular calcium mobilization compared to AT alone. Pre-treatment of the HUVEC with bradykinin (1 microM) for 2 minutes also inhibited the effects of alpha-thrombin (1 microM) on f-actin distribution examined by BODIPY-phallodin staining and increased the clotting times for an alpha-thrombin dependent fibrinogen to fibrin clotting assay. However, incubation of bradykinin (1 microM) with alpha-thrombin (1 microM) for either 10 minutes or 100 minutes produced no detectable hydrolysis products. These data strongly suggest that the inflammatory mediators alpha-thrombin and bradykinin when released together, rather than being synergistic, are antagonistic.

Actins↗

PubMed1997

Oxidized low-density lipoprotein increases endothelial intracellular calcium and alters cytoskeletal f-actin distribution.

Central to the pathogenesis of atherosclerosis is an abnormally functioning endothelium and a consequent loss of vascular integrity. These abnormalities may be induced by haemodynamic factors, biochemical substances, and also by oxidatively modified low-density lipoprotein (LDL). To understand the mechanism by which oxidized LDL causes endothelial dysfunction, human umbilical vein endothelial cells (HUVECs) were loaded with FURA-2, and intracellular calcium mobilization was studied in acute (seconds after LDL was injected) or chronic (24 h after LDL was injected) preparations. Our results demonstrate that 100 microg mL(-1) oxidized LDL increases HUVEC intracellular calcium. In contrast, native LDL at this same concentration had no effect. In addition, chronic exposure (24 h) of HUVECs to oxidized LDL significantly increases HUVEC intracellular calcium. Fluorescent photomicrographs of HUVECs stained with BODIPY-phalloidin f-actin indicates that oxidized LDL causes a reorganization of microfilaments. The results of this study demonstrate that the mechanism by which oxidized LDL causes a loss of vascular integrity could be through activation of endothelial cells to increase cytosolic calcium, which alters the endothelial barrier by reorganizing the cytoskeleton.

Actins↗

PubMed1997

Von Willebrand factor restores impaired platelet thrombogenesis in copper-deficient rats.

Dietary copper restriction reduces microvascular thrombogenesis. We have now examined the roles of shear forces and von Willebrand factor (vWF) in in vivo thrombus formation in the cremaster microcirculation of copper-deficient rats. Male weanling Sprague-Dawley rats were fed purified diets that were either copper-adequate (6.3 mg Cu/kg) or copper-deficient (0.3 mg Cu/kg) for 4 wk. Intravascular fluorescein isothiocyanate tagged to bovine serum albumin was activated with 450-490 nm light to induce thrombus formation in microvessels. Thrombus initiation time was significantly prolonged in copper-deficient rats; after thrombus appearance, however, vessel occlusion was significantly accelerated. The greater shear rates of arterioles compared with venules significantly increased the thrombus initiation time in both groups. However, vessel occlusion time and thrombus growth time were independent of shear rate. Intravascular vWF (0.2 u/100 g body wt) decreased thrombus initiation time in the CuD group without affecting thrombus growth time. The data suggest that decreased thrombogenesis in copper-deficient rats is not a result of altered rheological factors or arteriolar-venular differences, but appears to result from decreased platelet-to-endothelial cell adhesion.

Analysis of Variance↗

PubMed1997

Interleukin-2-induced hepatic injury involves temporal patterns of cell adhesion in the microcirculation.

The treatment of metastatic cancer with interleukin-2 (IL-2) is limited by systemic toxicities, including hepatic dysfunction. The objective of this study was to determine the cellular mechanisms of IL-2-induced hepatic injury. Intravital microscopy was used for the direct observation of the murine hepatic microcirculation after 2 h, 2 days, and 4 days of IL-2 treatment. At each interval, leukocyte- and platelet-endothelial adherence were observed and quantitated. Simultaneously, sinusoidal perfusion, serum levels of glutamate pyruvate transaminase, and edema were measured as indexes of hepatic toxicity. IL-2 increased neutrophil adhesion acutely in association with decreased sinusoidal perfusion. Leukocyte adhesion subsided at 2 days, but platelet-endothelial interactions were enhanced and 40% of mice receiving IL-2 had microvascular thrombi. These effects occurred in conjunction with decreased sinusoidal perfusion and the development of hepatic edema. After 4 days of IL-2, maximal hepatic edema, hypoperfusion, and increased serum levels of glutamate pyruvate transaminase were associated with increased lymphocyte adhesion and microvascular thrombosis. These data suggest coordinated, temporal roles of leukocytes and platelets in the generation of IL-2-induced hepatic injury.

Animals↗

PubMed1997

Antithrombotic and vasorelaxant properties of a novel synthetic RGD peptide containing nitric oxide.

We have developed a novel synthetic peptide containing both the antiadhesive Arg-Gly-Asp (RGD) amino acid sequence and a nitric oxide (NO) moiety well known for its vasorelaxant properties. The main objective of this study was to determine whether this hybrid molecule is concurrently effective with regard to antithrombotic and vasorelaxation actions. Studies of in vitro platelet adhesion and of in vivo platelet thrombus formation in the rat demonstrated that the RGD-NO peptide increased the antithrombotic characteristics of the RGD peptide alone. The RGD-NO peptide also caused relaxation of rat aortic rings, while the RGD peptide did not induce relaxation. These characteristics of Ac-RGDC-SNO suggest that this or similar compounds may have potential as effective antithrombotic agents in coronary and peripheral artery disorders.

Animals↗

PubMed1996

The role of cytokines, adhesion molecules, and chemokines in interleukin-2-induced lymphocytic infiltration in C57BL/6 mice.

IL-2 mediates the regression of certain malignancies, but clinical use is limited because of associated toxicities, including parenchymal lymphocytic infiltration with multiple organ failure. Secondarily induced cytokines are important mediators of IL-2 toxicity and IL-2-induced lymphocyte-endothelial adherence and trafficking. The recently discovered C-C chemokines, RANTES (regulated on activation, normal T expressed and secreted) and macrophage inflammatory protein-1alpha, have also been implicated in lymphocytic migration. We hypothesized that IL-2 alters cytokine, C-C chemokine, and adhesion molecule expression in association with parenchymal lymphocytic infiltration. C57BL/6 mice were injected with 3x10(5) IU of IL-2 or 0.1 ml of 5% dextrose intraperitoneally every 8 h for 6 d, then killed. IL-2 induced massive lymphocytic infiltration in the liver and lung and moderate infiltration in the kidney in association with organ edema and dysfunction. Immunostaining showed increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in association with this organ-specific lymphocytic infiltration. Flow cytometry showed increased expression of the corresponding ligands (lymphocyte function-associated antigen-1 and very late antigen-4) on splenocytes. IL-2 increased TNF-alpha mRNA and protein expression in the liver. Organs infiltrated by lymphocytes had increased TNF-alpha mRNA, whereas RANTES mRNA was increased in all organs, regardless of lymphocytic infiltration. IL-2 toxicity involves organ-specific TNF-alpha and RANTES production with increased ICAM-1 and VCAM-1 expression as potential mechanisms facilitating lymphocytic infiltration and organ dysfunction.

Animals↗

PubMed1996

Mechanisms of interleukin-2-induced hepatic toxicity.

Interleukin 2 (IL-2) mediates the regression of metastatic cancer, but its clinical use is limited by associated toxicities including hepatic dysfunction. To determine the mechanism for IL-2-induced hepatic dysfunction, we hypothesized that IL-2 activation of Kupffer cells causes leukocyte-endothelial adhesion and decreases hepatic sinusoidal blood flow. C57BL/6 mice were given injections of latex particles and prepared for intravital hepatic microscopy 2 h after i.p. IL-2 administration. Liver tissue was also prepared to quantitate hepatic tumor necrosis factor (TNF) mRNA and processed for light and electron microscopy. Phagocytosing Kupffer cells and leukocytes adherent to the endothelium were counted, and surface sinusoidal blood flow was quantitated. Kupffer cell activity was quantitated as the ratio of phagocytosing Kupffer cells to sinusoidal blood flow. IL-2 significantly increased Kupffer cell activity (0.56 +/- 0.05 for controls versus 0.84 +/- 0.05 for IL-2), significantly caused leukocyte-endothelial adhesion (26.7 +/- 7.9 for controls versus 87.0 +/- 27.6 for IL-2, WBC/mm2 endothelial surface), and significantly decreased the number of sinusoids containing blood flow per microscopic field (6.66 +/- 0.15 for controls versus 5.79 +/- 0.13 for IL-2) without causing changes in systemic hemodynamic parameters. In IL-2 treated livers, light and electron microscopy showed the constriction of sinusoids associated with swollen or ruptured mitochondria, which was consistent with hypoxic deterioration near central venules. Adherent platelets, neutrophils, and lymphocytes within sinusoids and central venules were also observed. PCR revealed that IL-2 significantly induced TNF mRNA expression in the liver. These data suggest that IL-2 activates Kupffer cells in association with the release of monokines including TNF, which causes activation of circulating leukocytes as well as hepatic sinusoidal endothelial cells. The resultant leukocyte and platelet adhesion to the endothelium may then physically impede the sinusoidal microcirculation, resulting in microscopic areas of hepatic ischemia and explaining the mechanism of IL-2-induced hepatic dysfunction.

Animals↗

PubMed1996

Mechanisms of alpha-thrombin, histamine, and bradykinin induced endothelial permeability.

alpha-Thrombin, bradykinin, and histamine are endogenous mediators that increase endothelial permeability. We examined the mechanism by which these three vasoactive mediators could alter permeability to albumin of human umbilical vein endothelial cells (HUVEC). HUVEC were grown to confluence on Transwell membranes and we monitored the flux of fluorescein isothiocyanate-labeled human serum albumin across the membrane from the upper to lower chamber of the Transwell. Addition of alpha-thrombin, bradykinin, or histamine increased the permeability coefficient of the HUVEC monolayer. At 30 min the permeability coefficient for alpha-thrombin was 4.92 x 10(-6) cm/sec while histamine was 4.47 x 10(-6) cm/sec. Maximum changes in the permeability coefficient were about three-fold control baseline values (1.59 x 10(-6) cm/sec). There was also a temporal difference in the magnitude of the permeability coefficient. alpha-Thrombin and bradykinin induced HUVEC permeability was increased for the first 90 min after which it returned to control levels. In contrast, histamine increased the permeability of the HUVEC monolayer throughout the 2 h experiment. To determine a possible intracellular mechanism of the altered permeability coefficients, HUVEC were labeled with FURA-2 and intracellular calcium was monitored by digital fluorescence ratio imaging. Maximum intracellular calcium in HUVEC was increased by alpha-thrombin (245 +/- 20 nM) and histamine (210 +/- 22 nM), but not by bradykinin (70 +/- 7 nM) as compared to control (69 +/- 10). Fluorescent photomicrographs of HUVEC stimulated with the three agonists indicated that alpha-thrombin and histamine substantially altered HUVEC f-actin arrangement, while bradykinin had no effect on HUVEC f-actin distribution. These data support previous in vitro and in vivo studies demonstrating increased permeability by all three agonists. These data also show, for the first time, that histamine and alpha-thrombin increased permeability by calcium-dependent intracellular pathways, but bradykinin operates through a calcium-independent mechanism.

Actins↗

PubMed1996

Interleukin-10 inhibits interleukin-2-induced tumor necrosis factor production but does not reduce toxicity in C3H/HeN mice.

Immunotherapy with interleukin-2 (IL-2) is limited by severe side effects thought to be mediated by the activation of immune effector cells and the induction of secondary cytokines including tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). In C3H/HeN mice the primary IL-2 toxicity is the production of pleural effusion with subsequent respiratory compromise. IL-10 is a cytokine that has been shown to inhibit the generation of secondary cytokines in vitro and in vivo. In this study, in C3H/HeN mice, we tested the ability of IL-10 to inhibit IL-2-induced mononuclear cell and alveolar macrophage activation and IL-2-induced increases in serum TNF-alpha and IFN-gamma, all of which may contribute to the generation of toxicity. IL-10 was ineffective at reducing IL-2-induced pleural effusion. However, IL-10 did inhibit IL-2-induced increases in serum TNF-alpha, which was accompanied by a decrease in Golgi apparatus and rough endoplasmic reticulum in alveolar macrophages. In addition, IL-10 combined with IL-2 increased mononuclear cell activation, which may limit the ability of IL-10 to inhibit IL-2-induced IFN-gamma production and pulmonary injury.

Animals↗

PubMed1996

Microvascular responses in the skeletal muscle of the diabetic rat.

We examined microvascular responses to acetylcholine (ACh), adenosine (ADO), nitroprusside (SNP), bradykinin (BK), histamine (HIS), and serotonin (5-HT) in control and diabetic rats. Agonists were applied topically to neurovascularly intact and environmentally controlled cremaster muscles of diabetic or control rats 3 weeks after streptozotocin or vehicle injection. Precapillary arteriolar diameters and leakage of fluorescein isothiocyanate conjugated to bovine serum albumin (FITC-BSA) in postcapillary venules were measured by using intravital microscopy techniques. All agents dilated the arterioles, and BK, HIS, and 5-HT caused concentration-dependent leakage of FITC-BSA. Of the vasodilators tested, only ACh-induced dilation was attenuated in diabetic animals. BK and HIS caused a concentration-dependent leakage of FITC-BSA that was similar in diabetic and control groups, but FITC-BSA leakage to 5-HT was significantly attenuated in diabetic animals. To determine whether the attenuated responses were attributable to an alteration of nitric oxide, the same dilational and leakage-promoting agents were tested in normoglycemic animals in the presence of N'-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase. Only ACh-induced dilation in control animals was attenuated in the presence of L-NAME; all other responses were normal. Thus, although the attenuated ACh-induced arteriolar dilation in striated muscle of diabetic rats may be linked to an impairment of nitric oxide function or release, the reduced leakage to serotonin is not linked to nitric oxide. The impaired leakage response to 5-HT is specific for that agonist and is not an indication of a generalized decrease in vascular permeability in these diabetic animals.

Acetylcholine↗

PubMed1996

Interleukin-2 induces increased platelet-endothelium interactions: a potential mechanism of toxicity.

Cancer immunotherapy with interleukin-2 (IL-2) is limited by side effects that may cause organ dysfunction. The role of platelets in the generation of IL-2-induced organ dysfunction has not been studied, although various studies have shown that IL-2 therapy activates both platelets and the vascular endothelium. We hypothesized that IL-2 therapy may enhance the thrombogenic response to inflammatory stimuli through increased platelet-endothelial interactions and that these effects could lead to the development of organ dysfunction. C57BI/6 mice were treated with IL-2 intraperitoneally for 2 hours (short term) or 2 to 5 days (long term) and prepared for in vivo microscopy of the ear microcirculation. Mice were injected intra-arterially with fluorescein isothiocyanate conjugated to bovine serum albumin (FITC-BSA). Blue light activation of the FITC-BSA in ear arterioles induced thrombus formation. The time to initial thrombus formation was measured as an index of thrombogenicity. Platelet function was analyzed by aggregometry and platelet expression of IL-2 receptors, and the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) was analyzed by flow cytometry. Organ dysfunction was evaluated by serum markers. The administration of both short-term and long-term IL-2 reduced the time to initial thrombus formation as compared with controls. In vitro platelet aggregometry revealed no acute alterations in platelet function; however, long-term IL-2 treatment resulted in decreased disaggregation rates. There were no platelet IL-2 receptors present, and the expression of the adhesion molecule LFA-1 was not altered by IL-2. Increased thrombogenicity occurred before the generation of organ dysfunction. These data suggest that increased platelet adherence induced by IL-2 is caused by effects on the endothelium that could result in microvascular thrombus formation and contribute to organ dysfunction.

Adenosine Diphosphate↗

PubMed1996

Oxidized low density lipoprotein inhibits platelet plasma membrane Ca(2+)-ATPase.

Oxidized low density lipoprotein (LDL) has been shown to enhance platelet activation. Since platelet activation is accompanied by an increase in cytosolic calcium, the effects of oxidized LDL on plasma membrane Ca(2+)-ATPase, plasma membrane fluidity and cytoplasmic calcium were studied in human platelets and purified platelet plasma membranes. Our results demonstrate that oxidized LDL, but not native LDL, inhibits the activity of Ca(2+)-ATPase in purified platelet plasma membranes (P < 0.01). Addition of the free radical scavenger alpha-tocopherol had no effect on the ability of oxidized LDL to inhibit the Ca(2+)-ATPase. An increased cytoplasmic calcium level in whole platelets was induced by oxidized LDL (P < 0.01), indicating that the plasma membrane Ca(2+)-extrusion pump may also be inhibited in vivo by oxidized LDL, although other mechanisms for the increase in cytoplasmic calcium are possible. Since no change in membrane fluidity was observed in platelet plasma membranes exposed to oxidized or native LDL as estimated by steady state trimethylammonium diphenylhexatriene (TMA-DPH) anisotropy, oxidized LDL does not affect the Ca(2+)-ATPase by grossly changing the membrane environment. The present results suggest that exposure of platelets to oxidized LDL causes inhibition of the plasma membrane Ca(2+)-ATPase which contributes to the observed increase in cytoplasmic calcium and increased sensitivity to agonists.

Blood Platelets↗

PubMed1996

Platelet aggregation and adhesion during dietary copper deficiency in rats.

The role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 micrograms copper/g of diet) or copper-deficient (CuD, 0.3 microgram/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rate endothelial cells. After a one hour incubation at 37 degrees C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 x 10(-4)M) and measured in a turbidometric aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from ++CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.

Animals↗

PubMed1995

The microphotohemolytic response of erythrocytes is altered by streptozotocin-induced diabetes and copper deficiency in rats.

Microphotohemolysis is a new technique that has been used to determine the presence of alterations in the erythrocyte membrane. The method involves light activation through a microscope of a fluorescent dye-erythrocyte-buffer solution in a hemocytometer. The interaction of the light and dye result in the generation of toxic oxygen products which attack the membrane allowing water to enter the cell. As hemolysis occurs the optical density of the microscopic field decreases and this is recorded for later quantitation with an image analysis system. Maximal effect, time to half maximal effect and the slope of the hemolysis curve are determined. The goal of this study was to determine if microphotohemolysis could be used to detect differences in erythrocytes from animals with altered physiological states such as hypercholesterolemia, diabetes, and copper deficiency. These are conditions that alter the lipid or protein structure of the erythrocyte membrane and/or the antioxidative capacity of the erythrocyte. There were no effects of hypercholesterolemia on the microphotohemolytic response of the erythrocyte. Streptozotocin-induced diabetes resulted in a decreased maximum effect, a significant shift of the hemolysis curve to the right (increased T 1/2) and a significant decrease in the slope of the hemolysis curve. Copper deficiency resulted in a significant decrease in the slope of the hemolysis curve. These results in diabetes and copper deficiency are consistent with an altered protein structure in the erythrocyte membrane that occurs in these conditions. The data demonstrate that this technique may be used to detect differences between normal and altered erythrocytes. As such, it could be useful in monitoring the course of a disease or its treatment.

Animals↗