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Biomedical subjects

W Siess

Publications and source records attributed to W Siess.

At least 73 records · Page 4Linked to original sources
PubMed1985

Separation of inositol phosphates and glycerophosphoinositol phosphates by high-performance liquid chromatography.

We developed a HPLC method which separates the following nine inositol-containing compounds of biological interest: inositol, inositol 1-monophosphate, inositol 2- or 4-monophosphate, inositol 1,2-cyclic phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate, glycerophosphoinositol, glycerophosphoinositol 4-monophosphate, and glycerophosphoinositol 4,5-bisphosphate. The method shows good resolution and sufficient recovery (70-80%) for each compound. By applying this method to human platelets prelabeled with [3H]inositol and stimulated with thrombin, we found an early increase of inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate. Accumulation of glycerophosphoinositol, inositol 1-monophosphate, and an inositol monophosphate which cochromatographs with inositol 2- and inositol 4-monophosphate occurs later. The method is simple, and--after removal of salts from the incubation buffer--can be directly applied to the measurement of aqueous soluble [3H]inositol-labeled compounds in biological samples.

Chromatography, High Pressure Liquid↗
PubMed1985

A comparative study of eicosapentaenoic acid metabolism by human platelets in vivo and in vitro.

During long-term dietary n-3 fatty acid supplementation, eicosapentaenoic acid (EPA) is not incorporated into phosphatidylinositol or -serine of human platelets in vivo and is not detectable in phosphatidic acid upon stimulation with thrombin. However, EPA is released from platelet phospholipids and metabolized to thromboxane B3 (TXB3). In contrast, in vitro, platelets incorporate [14C]EPA into phosphatidylinositol, whether they contain endogenous EPA in their cellular lipids or not. Following platelet stimulation, [14C]EPA appears in phosphatidic acid, as free fatty acid, and is transformed to TXB3. We conclude that the fatty acid compositions of platelet phospholipid subclasses are regulated with a high degree of specificity in vivo. Qualitative differences exist between in vivo and in vitro uptake of EPA into platelet phospholipid subclasses. After in vivo incorporation, EPA is released by action of a phospholipase A2.

Adult↗
PubMed1985

Prostaglandin endoperoxide analogues stimulate phospholipase C and protein phosphorylation during platelet shape change.

We have studied the effects of two stable prostaglandin endoperoxide analogues on platelet lipid metabolism in relation to specific platelet functional changes. During platelet shape change, the endoperoxide analogues induce the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of a phosphoinositide-specific phospholipase C. In parallel, they stimulate the phosphorylation of a 40-kd and a 20-kd protein. During platelet shape change, arachidonic acid is released, but not metabolized by platelet cyclo-oxygenase or lipoxygenase. Phospholipase C activation and platelet shape change are independent of extracellular Ca++ and Mg++, arachidonate metabolism, and release of adenosine diphosphate (ADP). Activation of phospholipase C during platelet aggregation seems, however, to be mediated partly by release of ADP. We conclude that endoperoxide analogues initially stimulate in platelets the formation of products derived from phospholipase C activation, which might serve as intracellular messengers for phosphorylation of specific proteins related to platelet shape change.

Adenosine Diphosphate↗
PubMed1984

Dihomogammalinolenic acid, but not eicosapentaenoic acid, activates washed human platelets.

Dihomogammalinolenic acid (2.5-20 microM) added to suspensions of washed human platelets induces platelet shape change and the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of phospholipase C. It also stimulates the phosphorylation of a 40 kDa protein, indicating the activation of protein kinase C. Dihomogammalinolenic acid is converted mainly to 12-hydroxyheptadecadienoic acid and to a smaller extent to prostaglandin E1 and thromboxane B1. Small quantities of the lipoxygenase product 12-hydroxyeicosatrienoic acid are also observed. Indomethacin, by blocking platelet cyclooxygenase, prevents the activation of phospholipase C, protein kinase C, and platelet shape change induced by dihomogammalinolenic acid. Compound UK 38485, a specific thromboxane synthetase inhibitor, does not block platelet activation induced by dihomogammalinolenic acid. The results indicate that endoperoxides derived from dihomogammalinolenic acid, such as prostaglandin G1 or prostaglandin H1, may be responsible for the stimulation of phospholipase C and protein kinase C, and for the induction of platelet shape change. Eicosapentaenoic acid does not activate platelets and is poorly metabolized by platelet cyclooxygenase and lipoxygenase. Eicosapentaenoic acid is a better inhibitor of platelet activation induced by various agonists in washed platelets than dihomogammalinolenic acid. Eicosapentaenoic acid and dihomogammalinolenic acid are, however, equally effective in inhibiting aggregation induced by collagen in platelet-rich plasma. We suggest that eicosapentaenoic acid might be a better antithrombotic agent than dihomogammalinolenic acid.

8,11,14-Eicosatrienoic Acid↗
PubMed1984

Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change.

The present study compares the molecular mechanism by which thrombin, platelet-activating factor, and epinephrine induce platelet activation. Thrombin and platelet-activating factor induce an initial activation of phospholipase C, as measured by formation of 1,2-diacylglycerol and phosphatidic acid, during platelet shape change which is independent of and dissociated from metabolism of arachidonic acid. Phospholipase C activation and shape change are independent of extracellular Ca2+ and Mg2+. Formation of cyclooxygenase products occurs subsequent to the initial activation of phospholipase C and those metabolites are associated with platelet aggregation and further activation of phospholipase C. On the other hand, epinephrine is an unique platelet stimulus since it requires extracellular divalent cations and does not induce platelet shape change or activation of phospholipase C. Our results indicate that activation of phospholipase C may be a mechanism by which physiological agonists can activate platelets independently of extracellular divalent cations.

Arachidonic Acid↗
PubMed1984

Uptake, release and metabolism of docosahexaenoic acid (DHA, c22:6 omega 3) in human platelets and neutrophils.

Exogenous DHA is converted by human platelets to 14- and 11- HDHE and by human neutrophils mainly to 7- HDHE . Human platelets prelabeled with 14C-DHA, 14C-EPA and 14C-AA and stimulated with thrombin release and metabolize DHA only in trace amounts as compared to EPA and AA. 14C-DHA is incorporated into the 2-position of platelet phospholipids and occurs predominantly in phosphatidylethanolamine. DHA and EPA were also incorporated by dietary means into phospholipids of platelets and neutrophils. In resting platelets free DHA as well as free AA and EPA are not detectable. In platelets stimulated ex vivo with thrombin DHA is not significantly released which is in contrast to EPA and AA. After stimulation, 14- HDHE is found only in trace amounts as compared to 12-HETE and 12- HEPE . In DHA enriched neutrophils formation of HDHEs cannot be demonstrated after stimulation with ionophore A 23187. We conclude that even after dietary enrichment of DHA in phospholipids of platelets and neutrophils the level of free DHA and/or formation of HDHEs might be too low to substantially affect arachidonic acid metabolism and related functions of these cells.

Adult↗
PubMed1983

Arachidonic acid stimulates the formation of 1,2-diacylglycerol and phosphatidic acid in human platelets. Degree of phospholipase C activation correlates with protein phosphorylation, platelet shape change, serotonin release, and aggregation.

Exogenous unlabeled arachidonic acid (AA) added to human platelets prelabeled with [3H]AA induces breakdown of [3H]phosphatidylinositol and the rapid and transient formation of [3H]1,2-diacylglycerol and [3H]phosphatidic acid (PA), indicating activation of phosphatidylinositol-specific phospholipase C. Formation of [3H]1,2-diacylglycerol and [3H]PA is inhibited by pretreatment of platelets with aspirin, which suggests that endoperoxides or thromboxane A2 are responsible for AA-induced stimulation of phospholipase C. Exogenous unlabeled AA also induces the formation of [32P]PA or [14C]PA in platelets that have been prelabeled with 32Pi or [14C]AA, respectively. Increased radioactivity in PA reflects increased content of PA as measured by the fatty acid composition of PA. The relation of PA production, which reflects stimulation of phospholipase C, to specific platelet responses was further investigated. Low concentrations of AA (0.05-0.2 microM) induces platelet shape change in parallel to formation of 50-100% PA and phosphorylation of a 40,000 molecular weight protein. Higher concentrations of AA (0.5-50 microM) stimulate the formation of a further amount of PA (200-250%), and phosphorylation of 40,000 molecular weight protein, platelet aggregation, and serotonin release. Indomethacin inhibits all these observed changes by inhibiting the conversion of AA by platelet cyclooxygenase. In contrast, prostacyclin blocks these responses without affecting conversion of AA by platelet cyclooxygenase and thromboxane synthetase. We conclude that formation of endoperoxides and thromboxane A2 is necessary but not sufficient for platelet activation by AA. Only if PA is formed are platelets activated. The results indicate a central role for the phospholipase C pathway in the process of platelet activation.

Arachidonic Acid↗
PubMed1983

The role of phospholipase C in platelet responses.

Degradation of inositides induced by phospholipase C in activated platelets leads to the formation of 1,2-diacylglycerol (1,2-DG) and its phosphorylated product, phosphatidic acid (PA). We have studied the relationship between activation of phospholipase C and the appearance of specific platelet responses, such as phosphorylation of proteins, shape change, release reaction and aggregation induced by different stimuli such as thrombin, platelet-activating factor, collagen, arachidonic acid (AA) and dihomogamma linolenic acid. A low degree of platelet activation induces only shape change which is associated with partial activation of phospholipase C (formation of phosphatidic acid), and phosphorylation of both a 40K molecular weight protein (protein kinase C activation) and a 20K molecular weight protein (myosin light chain). A higher degree of platelet activation induces aggregation, release of serotonin and a higher level of phospholipase C and protein kinase C activities. Metabolism of AA occurs concomitantly to aggregation and serotonin release, but AA metabolites are not related to the shape change of human platelets. Platelet shape change and the initial activation of phospholipase C induced by thrombin or platelet-activating factor is independent of the metabolites derived from cyclo-oxygenase activity. Further activation of phospholipase C which occurs during platelet aggregation and release reaction is, however, partly dependent on cyclo-oxygenase metabolites.

Arachidonic Acid↗
PubMed1983

Properties and distribution of phosphatidylinositol-specific phospholipase C in human and horse platelets.

Phospholipase C has been studied in homogenates, total particulate and soluble fractions of horse and human platelets. This enzyme, assayed with exogenous L-3-phosphatidyl[14C]inositol, is predominantly localized in the soluble fraction and its distribution parallels that of lactate dehydrogenase. A small percentage of activity present in the particulate fraction seems to be due to contamination with soluble enzyme. Enzyme from horse and human platelets appears identical, having a Km of 0.10-0.15 mM, acid pH optimum (pH 5.5) and showing Ca2+-dependency and weak inhibition by deoxycholate. Analysis of the reaction products shows the formation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate in almost equal amounts. Platelet stimulation with thrombin does not seem to induce association of the cytosolic activity to the membranes. The cytosolic activity is not affected by pretreatment of the intact platelets with prostacyclin or thrombin. Degradation of phosphatidylinositol present in a membrane fraction isolated from platelets by cytosolic phospholipase C requires addition of deoxycholate. Our information suggests that the degradation of phosphatidylinositol in stimulated platelets is mainly achieved by exposure of the substrate to the cytosolic enzyme and by an increase of the free Ca2+ concentration needed for optimal phospholipase C activity.

Animals↗
PubMed1983

A role for cyclooxygenase products in the formation of phosphatidic acid in stimulated human platelets. Differential mechanisms of action of thrombin and collagen.

Human platelets prelabeled with (32P)orthophosphate or [14C]arachidonic acid (AA) were stimulated with collagen or thrombin, and platelet activation (shape change, aggregation, and release of serotonin) was determined in parallel to the formation of 32P- or 14C-labeled phosphatidic acid (PA). The results show a close correlation between the degree of platelet activation and the amount of PA formed. Activation of platelets and formation of PA induced by collagen (2 to 20 micrograms/ml) was blocked by pretreatment of platelets with trifluoperazine, indomethacin, aspirin, or N-methylimidazole. This suggests that the formation of AA by phospholipase A2 and its subsequent metabolism by cyclooxygenase and thromboxane synthetase are required for the collagen-induced formation of PA. Endoperoxide analog U-44069 induces formation of PA in human platelets that have been pretreated with or without aspirin. The action of thrombin does not follow the same pattern of collagen. Low concentrations of thrombin (0.05 units/ml) induce only platelet shape change and a small stimulation of PA, changes which are only minimally inhibited by indomethacin. However, a small increase in the thrombin concentration (to 0.1 unit/ml) induces platelet aggregation, release of serotonin, and a sharp increase in PA accumulation which are effectively inhibited by indomethacin. Even higher thrombin concentrations (0.4 to 0.8 units/ml), however, result in a further stimulation of PA formation, platelet aggregation, and release of serotonin which are insensitive to inhibition by indomethacin. The data show that cyclooxygenase metabolites of AA, produced after platelet activation, may be differentially involved in the formation of PA in platelets stimulated with collagen or thrombin. Formation of PA following collagen or intermediate concentrations of thrombin (0.1 to 0.2 units/ml) is dependent on the cyclooxygenase pathway. However, formation of PA by very low or by high concentrations of thrombin is not mediated by cyclooxygenase metabolites of AA.

Blood Platelets↗
PubMed1983

Effects of propranolol in vitro and in vivo on platelet function and thromboxane formation in normal volunteers.

In vitro and ex vivo effects of propranolol on platelet aggregation, formation of thromboxane B2 (TXB2) and platelet sensitivity to prostacyclin were studied in healthy men. Propranolol, added in vitro to platelet rich plasma (PRP) inhibited platelet aggregation and TXB2 formation induced by ADP, 1-epinephrine, collagen and arachidonic acid. Concentrations of 20-100 microM propranolol were effective when ADP, 1-epinephrine and collagen were used as stimuli. Higher concentrations (250-500 microM) were needed to inhibit aggregation induced by arachidonic acid. Oral administration of propranolol either as a single dose (120 mg) or for one week (3 x 40 mg/day) did, however, not affect platelet aggregation, thromboxane formation and platelet sensitivity to prostacyclin. In addition, withdrawal of propranolol was without effect on these parameters. Although propranolol has potent effects on platelet function in vitro, it seems that the blood levels achievable by oral administration of propranolol are too low to affect platelet aggregation and TXB2 formation.

Adult↗
PubMed1982

Arachidonic acid metabolites, hypertension and arteriosclerosis.

The level of arterial blood pressure is set by complete interactions of several mechanisms which influence both blood flow in and resistance of the vascular system. An imbalance favouring elevation of vascular resistance or extracellular volume will result in hypertension. Such alterations may include increased activity of the sympathetic nervous system, of the renin-angiotensin system, or excessive secretion of mineralocorticoids. Of equal importance may be a reduced activity of blood pressure-lowering factors such as prostaglandins and the kallikrein-kinin system. This paper describes the possible significance of prostaglandins in the pathophysiology of hypertension and in degenerative vascular disease, based on their involvement in the control of vascular resistance, renal regulation of extracellular volume and platelet-vessel wall interactions. An abnormality in the biosyn-thesis of certain prostaglandin endoperoxide metabolites may lead to hypertension even without an increase in the activity of the classic blood-pressure-elevating systems. The contribution of prostaglandins for the development of hypertension and degenerative vascular disease may be based on an inherent abnormality of the prostaglandin system, as well as on the effects of major risk factors such as dietary intake of sodium and fat on prostaglandin synthesis. Specific blockade or stimulation of distinct biosynthetic pathways leading to antagonistically acting prostaglandins and nutritional manipulation of precursor fatty acids should lead to a better understanding of the pathomechanisms involved and may offer new strategies for therapy or prevention of these cardiovascular disorders.

Arachidonic Acids↗
PubMed1982

In vitro prostaglandin synthesis by various rat renal preparations.

Prostaglandin synthesis by eight different structures from the rat kidney (while cortex, cortical tubules, glomeruli, outer medulla, papilla, glomerular cultured epithelial and mesangial cells, cultured interstitial medullary cells) was measured in vitro after incubation with [14C] arachidonic acid using high-performance liquid chromatography followed by RIA with four specific anti-prostaglandin antibodies (prostaglandin E2, prostaglandin F2 alpha, 6 keto-prostaglandin F1 alpha, thromboxane B2). Prostaglandin production by the whole cortex and cortical tubules was very low. The order of abundance for isolated glomeruli was thromboxane B2 great than prostaglandin E2 greater than prostaglandin F2 alpha greater than 6 keto-prostaglandin F1 alpha. Mesangial cells synthesized prostaglandin E2 at a markedly high rate, in decreasing order: prostaglandin F2 alpha, thromboxane B2 and 6 keto-prostaglandin F1 alpha. The same order of abundance was observed for epithelial cells. The papilla synthesized essentially prostaglandin E2 and prostaglandin F2 alpha, whereas the main product for the outer medullar was 6 keto-prostaglandin F1 alpha. Cultured interstitial cells synthesized mainly prostaglandin E2 and to a lesser extent prostaglandin F2 alpha. Unidentified peaks eluting between 6 keto-prostaglandin F1 alpha and thromboxane B2 were also observed chiefly with glomeruli but they were absent with the medullary preparations. They disappeared after incubation with indomethacin or aspirin and represented for glomeruli the greatest percentage of conversion of [14C] arachidonic acid. These results show that the prostanoid profile varies markedly with the different regions and cells of the rat kidney.

Animals↗
PubMed1982

Analysis of 6-keto-prostaglandin F1 alpha in human urine: age-specific differences.

A radioimmunoassay (RIA) for the estimation of 6-keto-PGF1 alpha in human urine is described in detail. The RIA method was validated by direct comparison to gas chromatography-mass spectrometry. In adults and in one year old children basal excretion of 6-keto-PGF1 alpha was found to be lower than that reported for PGE2 or PGF2 alpha. However, during the first week of life, significantly more 6-keto-PGF1 alpha was excreted. The very high levels of 6-keto-PGF in urine seen on the third day of life seemed already to decrease during the first week of life. It is concluded that prostacyclin may have a major role for kidney function in the newborn, possibly by protecting the immature kidney from high levels of angiotensin II.

6-Ketoprostaglandin F1 alpha↗
PubMed1982

Cytochalasins inhibit arachidonic acid metabolism in thrombin-stimulated platelets.

Low concentrations (0.5-1 microM) of cytochalasins inhibit the thrombin-stimulated polymerization of monomeric actin to filamentous actin in platelets. Similar concentrations of cytochalasin B inhibit the formation and metabolism of arachidonic acid in horse platelets stimulated by low concentrations of thrombin (0.1-0.5 unit/ml). However, the release of serotonin is not inhibited by cytochalasin B. Cytochalasins B and D (0.5-1 microM) markedly reduce, in thrombin-stimulated human or horse platelets, the metabolism of the liberated arachidonic acid by cyclooxygenase activity to thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid and the conversion of arachidonic acid by lipoxygenase activity to 12-hydroxy-5,8,10,14-icosatetraenoic acid. The generation of arachidonic acid from platelet phospholipids and the formation of phosphatidic acid are much less affected by cytochalasin B or D. Cytochalasins do not directly inhibit platelet cyclooxygenase, lipoxygenase, phospholipase A2, or phosphatidyl-inositol-specific phospholipase C. In addition, the metabolism of exogenously added arachidonic acid by intact platelets is not inhibited by cytochalasins B and D. The results indicate that polymerization of actin in platelets stimulated by thrombin may be required for the effective metabolism of arachidonic acid released from platelet phospholipids.

Actins↗