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Biomedical subjects

H Holmsen

Publications and source records attributed to H Holmsen.

At least 19 recordsLinked to original sources
PubMed1992

Chlorpromazine increases the turnover of metabolically active phosphoinositides and elevates the steady-state level of phosphatidylinositol-4-phosphate in human platelets.

Non-permeabilizing concentrations (< 40 microM) of chlorpromazine (CPZ) increase the radioactivity of phosphatidylinositol-4-phosphate (PIP) in platelets pre-labelled with [32P]Pi, but the biochemical mechanisms underlying this increase are poorly understood. Incubation of [32P]Pi-labelled, gel-filtered platelets with 25 microM CPZ for 10 min increased: (1) the mass of PIP from 315 to 476 nmol/10(11) platelets but not the total inositol phospholipid mass, (2) the specific phosphodiester radioactivities in phosphatidylinositol (PI), PIP and phosphatidylinositol-4,5-bisphosphate (PIP2) by 34, 63 and 37%, respectively, and (3) the specific phosphomonoester radioactivities in PIP and PIP2 by 53 and 10%, respectively. In control platelets (no CPZ) the specific radioactivity of the phosphodiester was the same in PI, PIP and PIP2, and the specific radioactivity in the phosphomonoester in PIP and PIP2 was 55% of that of the gamma-phosphoryl in ATP, measured as metabolically active, actin-bound ADP. These results suggest that 55% of each of PI, PIP and PIP2 constitutes a metabolic pool which is labelled by 32P in the platelets, while the remainder is in a metabolically inactive pool and not labelled. CPZ has two major effects: (1) CPZ interferes with the kinase and phosphohydrolase reactions that maintain the steady-state level of PIP in the metabolic phosphoinositide pool, resulting in a 92% increase in the PIP level of this pool, and (2) CPZ causes synthesis (45% in 10 min) of new phosphodiester in the metabolically active phosphoinositides by tentative stimulation of the turnover of the phosphoinositide cycle, de novo phosphoinositide synthesis and/or diacylglycerol formation through phospholipases C and D. The marked alteration by CPZ of phosphoinositide metabolism may be part of the mechanism by which this drug effects its psychotropic action.

Adenosine Diphosphate↗
PubMed1992

[From endothelium-derived relaxing factor to L-arginine and nitrogen monoxide].

Endothelium-derived relaxing factor has been identified as nitric oxide. It was originally found to be released from endothelial cells, mediating vascular relaxation and platelet inhibitory action. Nitric oxide is formed enzymatically from L-arginine by nitric oxide synthase, and this enzyme has up to the present been demonstrated in endothelial cells, platelets, neutrophils, macrophages, neurons and smooth muscle cells. Both a constitutive and an inducible nitric oxide synthase have been identified, which may serve different physiological/pathophysiological purposes. The biological implications of nitric oxide have now been manifested, and the number of reports on its physiological role is rapidly increasing. Compounds which inhibit the formation of nitric oxide have become available, and this expanding field has created great interest amongst clinicians.

Amino Acid Oxidoreductases↗
PubMed1992

Neomycin is a platelet-derived growth factor (PDGF) antagonist that allows discrimination of PDGF alpha- and beta-receptor signals in cells expressing both receptor types.

The aminoglycoside neomycin has recently been found to affect certain platelet-derived growth factor (PDGF) responses in C3H/10T1/2 C18 fibroblasts. Using porcine aortic endothelial cells transfected with PDGF alpha- or beta-receptors, we explored the possibility that neomycin interferes with the interaction between the different PDGF isoforms and their receptors. We found that neomycin (5 mM) inhibited the binding of 125I-PDGF-BB to the alpha-receptor with only partial effect on the binding of 125I-PDGF-AA; in contrast, the binding of 125I-PDGF-BB to the beta-receptor was not affected by the aminoglycoside. Scatchard analyses showed that neomycin (5 mM) decreased the number of binding sites for PDGF-BB on alpha-receptor-expressing cells by 87%. Together with cross-competition studies with 125I-labeled PDGF homodimers, the effect of neomycin indicates that PDGF-AA and PDGF-BB bind to both common and unique structures on the PDGF alpha-receptor. Neomycin specifically inhibited the autophosphorylation of the alpha-receptor by PDGF-BB, with less effect on the phosphorylation induced by PDGF-AA and no effect on the phosphorylation of the beta-receptor by PDGF-BB. Thus, neomycin is a PDGF isoform- and receptor-specific antagonist that provides a possibility to compare the signal transduction pathways of alpha- and beta-receptors in cells expressing both receptor types. This approach was used to show that activation of PDGF beta-receptors by PDGF-BB mediated a chemotactic response in human fibroblasts, whereas activation of alpha-receptors by the same ligand inhibited chemotaxis.

Aminoglycosides↗
PubMed1992

Glycerophospholipid metabolism: back to the future.

It has become customary to regard the various glycerophospholipids as quite similar, and the acyl groups are considered to have little influence on the behaviour of the lipids in membranes or metabolism. Nevertheless, a number of recent observations by the authors and others indicate a high degree of metabolic compartmentation and substrate specificity with regard to the acyl substituents (acyl specificity) of glycerophospholipid metabolising enzymes in intact cells. 1. [32P]Orthophosphate and [3H]glycerol are incorporated into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of platelets and Swiss 3T3 fibroblasts with a [32P]/[3H]-ratio several fold lower than in glycerol-3-phosphate, phosphatidic acid (PA) and phosphatidylinositol (PI), suggesting distinct metabolic separation (probably by cellular compartmentation) of the glycerol and choline (or ethanolamine) branches of de novo phospholipid biosynthesis. 2. In fibroblasts the [32P]/[3H]-ratio varied 50-fold among the molecular species of PC, PE, PI and PA, which indicates that the enzymes involved in these conversions have some degree of acyl specificity. 3. In vitro assays for lipid-converting enzymes employ detergents, which affect acyl specificity of the enzymes (lipid kinases) both by their chemical nature and concentrations. 4. Thrombin stimulation of platelets causes formation of a multitude of diacylglycerol (DAG) molecular species, but only one major molecular species of PA is formed indicating that the DAG kinase may have distinct acyl specificity in the intact cell. 5. However, this specificity could also result from the net reactions of DAG kinase(s) and PA phosphohydrolase(s), which would constitute an ATP-utilising, paired regulation of the molecular species of PA and the inositol lipids on one hand, and PC, PE phosphatidylserine and triacylglycerol on the other. These findings indicate a high complexity of glycerophospholipid metabolism and a distinct acyl specificity in intact cells that are not apparent from studies in vitro. A major challenge for future research in this area is to bridge the apparent discrepancy between in vivo and in vitro observations regarding glycerophospholipid metabolism, an endeavour that will require more knowledge about the physical chemistry of naturally occurring molecular species than is available today. The most prevailing appreciation of glycerophospholipids among biological scientists to-day is that they can be distinguished functionally, topographically and metabolically only by their head groups and that they form the bilayer in biological membranes. Most of us know that the fatty acid in the sn-2 position is unsaturated and have been indoctrinated that the higher the degree of unsaturation, the greater the fluidity of the membrane.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Absence of PDGF-induced, PKC-independent c-fos expression in a chemically transformed C3H/10T1/2 cell clone.

The effect of platelet-derived growth factor (PDGF) on c-fos mRNA transcription was studied in the immortalized mouse embryo fibroblast C3H/10T1/2 Cl 8 (10T1/2) cells and the chemically transformed, tumorigenic subclone C3H/10T1/2 Cl 16 (Cl 16). In the 10T1/2 cells as well as the Cl 16 subclone, the dose-dependent PDGF stimulation of c-fos mRNA synthesis was similar in both logarithmically growing and confluent cultures. c-fos mRNA was induced severalfold by 12-O-tetradecanoylphorbol-13-acetate (TPA) in both 10T1/2 and Cl 16. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment inhibited PDGF-stimulated c-fos mRNA expression in Cl 16 cells but did not affect this induction in the 10T1/2 cells. This inhibition was not a general phenomenon of 3-methylcholanthrene-mediated transformation of 10T1/2 cells since experiments with another transformed 10T1/2 cell clone, C3H/10T1/2 TPA 482, gave qualitatively the same results as the 10T1/2 cells. Receptor binding experiments showed that the nontransformed and transformed cells had a comparable number of PDGF receptors, 1.3 x 10(5) and 0.7 x 10(5) receptors per cell, respectively. Furthermore, cAMP-induced c-fos expression induced by forskolin is formerly shown to be independent of PKC down-regulation. In our experiments, forskolin induced c-fos expression in both clones. However, PKC down-regulation inhibited the forskolin-induced c-fos expression in Cl 16 cells. This apparently demonstrates cross talk between PKC and PKA in the c-fos induction pathway. The present results provide evidence for an impaired mechanism for activating c-fos expression through PKC-independent, PDGF-induced signal transduction in the chemically transformed Cl 16 fibroblasts compared to that in nontransformed 10T1/2 cells.

Adenosine Triphosphate↗
PubMed1992

Porcine platelets in vitro and in vivo studies: relevance to human thrombosis research.

This review summarizes present knowledge on porcine platelets in vitro and recent studies on in vivo activation of platelets in the pig. There are certain differences compared to human platelets: Platelet aggregation and secretion cannot be achieved by epinephrine, and the arachidonate pathway seems poorly developed in porcine platelets. Genetic models for von Willebrand disease (vWD) and storage pool deficiency (SPD) have been developed in the pig. Several models for the study of in vivo platelet deposition and early thrombus formation have been developed. Platelet radio-labeling techniques (with 111In) have been used extensively. We conclude that the pig seems to be a good choice for the investigation of in vivo platelet activation and deposition based on present knowledge of porcine platelets and on already established animal models.

Angioplasty, Balloon↗
PubMed1992

Effects of hyperoxia on human endothelial cell proliferation and morphology in vitro.

The effects of hyperoxia on endothelial cells from human umbilical veins were studied in vitro by flow cytometric DNA analysis, cell proliferation, and morphology. Exposure of the cells to PO2 of approximately 400 and 500 mmHg (corresponding to O2 concentrations of 65 and 95%, respectively) caused, after 24 h, an increase in the proportion of S-phase cells from 18 to 33%. This was followed by a dramatic decrease of S-phase cells to about 4% after 48 h of O2 exposure. This decrease was also sustained after 72 h of O2 treatment. Concomitant with the depletion of S-phase cells was an increase from 13 to 21% in the proportion of cells in the G2M phase, indicating that cells were unable to undergo cell division after a 48-h exposure to 65 and 95% O2. Cells exposed to various PO2 levels (124, 248, 400, and 500 mmHg) showed a dose-dependent reduction in cell proliferation leading to a total inhibition of cell growth after exposure to PO2 of 400 and 500 mmHg. Cells exposed to 248 mmHg PO2 exhibited similar growth rates as the control cells during the first 3 days. Thereafter there was a 50% reduction in cell density as compared to the control cells. Light and scanning electron microscopy showed that the hyperoxic cells underwent morphologic changes characterized by cell contraction and swelling. Thus it is shown that exposure to very high PO2 tensions first stimulates the cells to enter the S phase of the cell cycle. Thereafter changes take place leading to a nearly complete reduction of cell proliferation.

Cell Count↗
PubMed1992

Activation of blood platelets in workers exposed to organic solvents.

Organic solvents probably exert their clinical effects through distortion of cellular membrane structure and function. Blood platelets represent an easily available membrane system. This study describes an evaluation of mean platelet volume (MPV) and total platelet count (TPC) in a group of workers exposed to a mixture of organic solvents in paint factories. We have found a correlation between decreased MPV and present exposure. The reduction was reversed within an exposure-free interval of 3 weeks. The intracellular adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio was significantly increased in exposed workers, compatible with dense granule secretion. A quantitative study of the phosphoinositide cycle in the platelets revealed an increased concentration of the phosphatidic acid, indicating an increased turnover. These findings are consistent with a solvent-induced activation of circulating platelets.

Adenosine Diphosphate↗
PubMed1991

Microbubble-induced serotonin secretion in human platelets.

The effect of nitrogen (N2) microbubbles on platelets resembles that of common platelet agonists with respect to aggregation (Thorsen T et al., Undersea Biomed Res 1986; 13: 289-303). In the present study we examined the effect of microbubbles on platelet secretion of preloaded 14C-serotonin. We demonstrate that stirring of platelet-rich plasma with N2-microbubbles causes a loss of single platelets that is associated with secretion. However, secretion did not increase above baseline values until after 20 min of microbubble exposure, when platelet aggregation had reached 40%. After that time the secretion rate increased. There was no correlation between secreted serotonin and the degree of platelet aggregation. Although no 14C-serotonin secretion occurred in presence of acetylsalicyclic acid (ASA), microbubble-induced platelet aggregation was only marginally reduced. Epinephrine alone caused significant platelet aggregation but no 14C-serotonin secretion and it enhanced N2-microbubble-induced platelet aggregation and secretion; ASA completely prevented secretion under these circumstances but failed to abolish the enhancement of aggregation compared with microbubbles alone. Earlier studies have shown that platelets adhere to the bubble surfaces (Thorsen T et al., Undersea Biomed Res 1987; 14: 45-59). The results in the present study indicate that non-adhering platelets in the bulk phase are not activated by means of autocrine stimulation through dense granule material.

Blood Platelets↗
PubMed1991

Identity between palmitoyl-CoA synthetase and arachidonoyl-CoA synthetase in human platelet?

Apparent Km values have been determined for the substrates ATP, CoA and fatty acids for the long-chain acyl-CoA synthetase (EC 6.2.1.3) reaction in lysates of human blood platelets. The apparent Km for ATP was higher for saturated fatty acids (C12:0 to C18:0) than for unsaturated acids (C18:1 to C22:6). Other apparent Km values were very similar for all long-chain fatty acids tested. Palmitic acid inhibited the formation of [14C]arachidonoyl-CoA, and arachidonic acid inhibited the formation of [14C]palmitoyl-CoA, with [14C]arachidonate or [14C]palmitate respectively as substrate. After chromatography of Triton X-100-extracted platelet protein in several systems (hydroxyapatite, DEAE-Sepharose, Sephacryl S-200 HR, CoA-Sepharose, Sephadex G-100 and AcA 34), both arachidonoyl-CoA synthetase and palmitoyl-CoA synthetase activities were eluted together in the various protein peaks, and with approximately the same ratio of activities in all peaks. After some purification steps (DEAE-Sepharose and Sephacryl S-200 HR), the acyl-CoA synthetase activity was up to 37 nmol/min per mg of protein with [14C]palmitate as substrate, and up to 116 nmol/min per mg of protein with [14C]arachidonate as substrate. The purification was respectively about 8- and 10-fold. The results indicate that palmitoyl-CoA (or unspecific) synthetase and arachidonoyl-CoA (or specific) synthetase are in fact the same enzyme, in agreement with previously reported results from this laboratory.

Blood Platelets↗
PubMed1991

Stimulation of human platelets with low concentrations of thrombin: evidence for equimolar accumulation of inositol trisphosphates and phosphatidic acid.

1. Gel-filtered human platelets prelabeled with [32P]Pi or [3H]glycerol were exposed to 0-0.3 U/ml of thrombin and analyzed for radioactivities and masses in the phosphoinositides, inositol trisphosphates (IP3), phosphatidic acid (PA) and diacylglycerol (DAG) at 15 and 180 sec of stimulation. 2. At thrombin concentrations below 0.1 U/ml, PA and IP3 accumulated in equimolar amounts. 3. The production and disappearance of the metabolites of the polyphosphoinositide cycle was balanced during 180 sec of stimulation with 0.03-0.1 U/ml of thrombin. 4. Under these conditions no increase in [3H]DAG or [3H]monoacylglycerol could be detected. 5. The data indicate that all DAG is converted to PA and support our conclusion that phosphatidylinositol 4,5-bisphosphate represents the major source for production of DAG upon stimulation of human platelets with low concentrations of thrombin.

Blood Platelets↗
PubMed1991

Fatty acids in human platelets and plasma. Fish oils decrease sensitivity toward N2 microbubbles.

Platelet aggregation induced by N2 microbubbles (simulating microbubbles developed during deep diving) was measured in seven volunteers before and after intake of ethyl-eicosapentaenoate (-EPA, 3.5 g/day) and ethyl-docosahexaenoate (-DHA, 2.5 g/day) for 2 wk. The relative content of arachidonic acid (AA) decreased in platelets from all individuals, whereas the content of EPA and DHA increased. The decrease of AA was almost identical with the increase of EPA plus DHA. In plasma the AA content was unchanged, while EPA and DHA increased. The N2 microbubble-induced aggregation showed a significant negative correlation with the DHA content both in platelets and in plasma. Less aggregation was also observed with high EPA content in platelets or plasma. A significant correlation between AA content in platelets and aggregation was seen. Intake of marine oils may be beneficial to divers under deep diving and to patients during extracorporeal circulation, because this may reduce the microbubble-induced aggregation.

Adult↗
PubMed1991

Effect of local hemostatics on platelet aggregation.

The platelets play an important role in the normal hemostasis, and it is known that both natural and synthetic macromolecules may induce platelet activation and aggregation. Thus, the purpose of the present study was to investigate the platelet aggregating effect of five different local hemostatics. Platelet aggregation was assessed by aggregometry. Unwoven fleece of bovine collagen polymer in fibrillar form induced aggregation in combination with small amounts of platelet agonists; ADP and adrenaline. Ordinary, nonabsorbable bone wax also induced aggregation in combination with the agonists, but larger concentrations of agonists were needed. Bioerodible polyorthoester with physical properties such as bone wax, oxidized cellulose and gelatin sponge did not promote platelet aggregation.

Adenosine Diphosphate↗
PubMed1991

Signal transducing mechanisms in platelets.

Platelets respond through discrete receptors to a number of physiological stimuli and foreign surfaces with a sequence of measurable responses: shape change, aggregation, secretion and arachidonate liberation. Three secretory responses are distinguished: release of substances from 1) dense granules (ADP, serotonin), 2) alpha-granules (coagulation factors, platelet-specific proteins, adhesive proteins) and 3) lysosomes (acid hydrolases). The liberated arachidonate is converted to prostaglandins and thromboxanes which, together with secreted ADP and close cell contact, will cause further platelet activation through "positive feedback" (autocrine stimulation). Some agonists are "weak" (ADP, vasopressin, platelet-activating factor) and depend on positive feedback to promote the full sequence of responses, while other agonists are "strong" (thrombin, collagen) and stimulate the entire response sequence without positive feedback. Most agonists appear to stimulate platelet responses via G-protein-dependent activation of phospholipase C, resulting in diesteratic hydrolysis of phosphatidylinositol-4,5-bisphosphate yielding inositol-1,4,5-trisphosphate and diacylglycerol. These are signal molecules which mobilize cytoplasmic Ca2+ and stimulate protein kinase C, respectively. Cytoplasmic Ca2+ will in turn activate protein phosphorylations which eventually lead to execution of the various responses while activation of protein kinase C appears to be linked to regulation of intracellular pH through Na+/H+ exchanger and to termination of the Ca(2+)-mediated signal processing. Other agonists (prostaglandins I2 and D2) counteract platelet stimulation through classical activation of adenylate cyclase.

Animals↗
PubMed1990

Trifluoperazine enhances accumulation and inhibits phosphohydrolysis of phosphatidate in thrombin-stimulated platelets.

Trifluoperazine (TFP) in concentrations up to 10-15 microM increased the formation of phosphatidic acid (PA) in platelets treated with 0.5 U/ml of thrombin, while higher concentrations of TFP inhibited formation of PA. Liberation of arachidonate (AA) from platelet phospholipids was progressively inhibited as the concentration of TFP increased. At thrombin doses lower than 0.1 U/ml TFP, (less than or equal to 25 microM) enhanced PA formation with either no effect of AA liberation (6 donors) or with much greater enhancement of PA formation than the decrease in liberation of AA (3 donors). The enhancement of PA formation by TFP did therefore not seem to be due to inhibition of phospholipase A2 (PLA2) by the phenothiazine, which has been suggested. We show further that TFP inhibits PA phosphohydrolase in platelet lysates, although with complex kinetics. It is therefore concluded that the enhancement of thrombin-induced PA production by TFP is not caused by inhibition of PLA2 but could be due to TFP-induced inhibition of PA phosphohydrolase.

Arachidonic Acid↗
PubMed1990

Neomycin inhibits PDGF-induced IP3 formation and DNA synthesis but not PDGF-stimulated uptake of inorganic phosphate in C3H/10T1/2 fibroblasts.

Porcine PDGF was found to increase [3H]inositol trisphosphate, [3H]thymidine incorporation and 32P-labelling of polyphosphoinositides in C3H/10T1/2 Cl 8 fibroblasts. These responses to PDGF stimulation were all inhibited by 5 mM neomycin, a polycationic aminoglycoside formerly known to inhibit polyphosphoinositide turnover. PDGF also markedly increased the cellular uptake of inorganic [32P]Pi. This response of PDGF was not inhibited by neomycin (5 mM). Thus, neomycin inhibited PDGF-induced IP3 formation, 32P-labelling of polyphosphoinositides and DNA synthesis, but not cellular uptake of inorganic phosphate. These effects of neomycin suggest a bifurcation of the initial part of the PDGF-induced signal transduction, separating at the receptor level or before phospholipase C activation.

Animals↗
PubMed1990

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Animals↗