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K Romstedt

Publications and source records attributed to K Romstedt.

12 recordsLinked to original sources
PubMed1992

Synthesis and alpha-adrenergic activities of 2- and 4-substituted imidazoline and imidazole analogues.

Seven analogues of medetomidine and naphazoline were synthesized and evaluated for their alpha 1 (aorta) and alpha 2 (platelet) activities. The analogues were composed of 2- and 4-substituted imidazoles and imidazolines attached through a methylene bridge to either the 1- or 2-naphthalene ring system. In general the 1-naphthalene analogues were the most potent inhibitors of epinephrine-induced platelet aggregation. Of considerable interest was the fact that the 1-naphthalene analogues (2, 5-7) were partial agonists while the 2-naphthalene analogues (3, 8, 9) were antagonists in an alpha 1-adrenergic system (aorta). Thus, appropriately substituted naphthalene analogues of medetomidine and naphthazoline provide a spectrum of alpha 1-agonist, alpha 1-antagonist, and alpha 2-antagonist activity.

Adrenergic alpha-Agonists↗
PubMed1988

Human platelet activation by bacterial phospholipase C: mechanism of inhibition by flurazepam.

We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes platelet activation possibly by inducing turnover of phosphoinositides and phosphorylation of a 47,000 Dalton protein (P47). Moreover, only 15 microM and 11 microM flurazepam inhibits PLC-induced platelet aggregation and serotonin secretion by 50% respectively. This study was conducted to better understand the mechanism of platelet activation by PLC and its inhibition by flurazepam. Incubation of (14C)-arachidonic acid labelled platelets with PLC produced diacylglycerol in a time- and concentration-dependent manner. Flurazepam did not inhibit diacylglycerol production by PLC. Paranitrophenolphosphorylcholine and prostaglandin E1 inhibited diacylglycerol production by 75% and 20% respectively. In a platelet-free system PLC hydrolyzed 14C-choline-phosphatidylcholine (14C-PC) in a time- and calcium ions-dependent manner. Flurazepam had no effect on PLC-induced hydrolysis of 14C-PC. Platelet cytosolic fraction (PCF), containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed (3H-inositol)-phosphatidylinositol (3H-PI) in a platelet-free system. Flurazepam did not inhibit hydrolysis of 3H-PI by PCF. Phospholipase C caused phosphorylation of P47 in 32P-labelled platelets. Flurazepam did not block phosphorylation of P47 in the first three minutes and had very little inhibitory effect by five minutes. However, flurazepam completely blocked phosphorylation of P47 by seven minutes. Platelet aggregation induced by ionomycin, a calcium ionophore, was completely inhibited by 100 microM flurazepam whereas platelet aggregation induced by 12-O-Tetradecanoylphorbol-13-acetate (TPA), which mimics the action of diacylglycerol, was partially inhibited by 300 microM flurazepam. These findings suggest that PLC induced platelet activation depends, at least in part, on diacylglycerol production and phosphorylation of P47. These data also suggest that flurazepam does not inhibit PLC-induced platelet activation by inhibiting: (a) the production of diacylglycerol from phosphatidylcholine; and (b) the action of PI-PLC on phosphatidylinositol. The ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that flurazepam is able to block platelet activation by inhibiting the increase in free cytosolic calcium ions in platelets or by inhibiting a step subsequent to the rise in intraplatelet calcium ions.

Alprostadil↗
PubMed1987

Synthesis and platelet antiaggregatory activity of trimetoquinol analogs as endoperoxide/thromboxane A2 antagonists.

Trimetoquinol (TMQ) has activity as a beta-adrenergic agonist and as a platelet antiaggregatory agent. Recent reports from this and other laboratories have focused on the mechanism of inhibition of platelet function by TMQ and its analogs. Based on its competitive and stereoselective inhibition of thromboxane mimetic agents, TMQ was proposed as an endoperoxide/thromboxane A2 receptor antagonist; however, this mechanism has been questioned. A radiolabeled TMQ analog with high specific activity would aid in the elucidation of the actual mechanism of action. In the current research, modifications of the trimethoxy ring system of TMQ have been investigated. Replacement of one or two of the methoxy groups with iodine atoms leads to retention of platelet antiaggregatory activity and agonist blocking activity. Thus, these analogs have promise as potential radioligands since iodide exchange labeling can provide 125I-labeled compounds. Further, replacement of a methoxy group with either a nitro or amino functionality leads to decreased activity in platelet systems. These results suggest that the putative sites of interaction for the trimethoxy ring system of TMQ in platelet systems will tolerate large, lipid soluble groups but will not tolerate large changes in the electronic characteristics of the ring system.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1986

Comparative antiaggregatory activity in human platelets of a benzopyranone aci-reductone, clofibric acid, and a 2,3-dihydrobenzofuran analogue.

A synthetic method for the preparation of aci-reductone 6-chloro-3,4-dihydroxy-2H-1-benzopyran-2-one (3) from 5-chlorosalicylate is presented. In human platelets, the benzopyranone derivative 3, clofibric acid (1), and the 2,3-dihydrobenzofuran analogue 4 inhibited aggregation and serotonin secretory responses to adenosine diphosphate (ADP) with a rank order of potency 3 greater than or equal to 4 greater than 1. Only analogues 3 and 4 consistently blocked the aggregatory responses (greater than 50%) to arachidonic acid (AA) and U46619, a thromboxane A2 agonist. Further, the rank order of inhibitory potency against U46619-induced serotonin secretion was 4 greater than 3 greater than 1. Benzopyranone 3 is of interest since it was the most potent inhibitor of thrombin-induced [3H]AA release (3 much greater than 4 = 1) and more potent than 1 or 4 for the blockade of the ADP- or AA-mediated pathway of platelet aggregation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1985

Beclobrinic acid--a new hypolipidemic agent--inhibits in vitro human platelet activation by blocking prostaglandin synthesis.

Effects and the mechanism of the antiplatelet actions of beclobrinic acid, free acid form of a new hypolipidemic agent beclobrate [(+)-2-[d-(P-chlorophenyl)p-tolyl)oxy)-2-methyl-butyrate), were examined using human platelets. Platelet-rich plasma (PRP) which has been prelabeled with (14C)-serotonin was incubated with beclobrinic acid (BBA) for one minute before the addition of various agonists. BBA (0.1-1.5 mM) inhibited platelet aggregation and serotonin secretion induced by ADP, epinephrine, arachidonic acid and collagen in a concentration dependent manner. BBA also inhibited arachidonic acid-induced production of malondialdehyde (MDA), a byproduct of prostaglandins, in a concentration dependent manner. However, up to 1.0 mM BBA did not inhibit platelet aggregation induced by U46619, a stable analog of prostaglandin H2. In other experiments BBA also blocked thrombin-induced release of (3H)-arachidonic acid from platelet phospholipids. These findings suggest that: (a) BBA inhibits platelet aggregation and serotonin secretion by inhibiting prostaglandin synthesis at two steps. First by interfering in the release of arachidonic acid from platelet phospholipids and second by inhibiting its conversion into prostaglandins; and (b) BBA does not inhibit the action of prostaglandins on human platelets.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1985

Benzodiazepines inhibit human platelet activation: comparison of the mechanism of antiplatelet actions of flurazepam and diazepam.

These studies were undertaken to examine the effects and the mechanism of action of flurazepam and diazepam on human platelet activation. One minute preincubation with flurazepam (3-300 microM) or diazepam (3-300 microM) inhibited platelet aggregation, serotonin secretion and prostaglandin synthesis induced by ADP (1-5 microM), epinephrine (1-5 microM), and arachidonic acid (600-1000 microM). However, 357% higher concentration of diazepam (265 microM) as compared to flurazepam (58 microM), was required to inhibit arachidonic acid induced production of malondialdehyde (MDA) by 50%. In addition, flurazepam and not diazepam inhibited the release of arachidonic acid from platelet phospholipids in a concentration dependent manner. In other experiments flurazepam but not diazepam also blocked aggregation and secretion induced by U46619 (2 microM), a stable analog of prostaglandin H2. Platelet aggregation and serotonin secretion induced by collagen (40-300 micrograms/ml) was inhibited by flurazepam with an IC-50 of 153 microM and 136 microM respectively, whereas higher than 300 microM diazepam was required to inhibit collagen-induced aggregation and secretion by 50%. Flurazepam and diazepam both exhibited their most potent antiplatelet effects against phospholipase C-induced aggregation which is mediated by prostaglandin-independent mechanisms. Only 15 microM and 11 microM flurazepam and 31 microM and 27 microM diazepam were needed to inhibit PLC-induced aggregation and secretion of serotonin by 50% respectively. Effects of these benzodiazepines on platelet cyclic AMP and cyclic GMP were also examined. Neither flurazepam nor diazepam caused any significant change in cyclic AMP or cyclic GMP levels in platelets. These findings suggest that: (a) flurazepam, as compared to diazepam, is 106% - 357% more effective in inhibiting platelet aggregation and serotonin secretion induced by arachidonic acid, collagen and phospholipase C; (b) flurazepam inhibits platelet activation by inhibiting the release of arachidonic acid, its conversion into prostaglandins and by blocking the action of prostaglandins on platelets; (c) diazepam does not inhibit thrombin-induced release of arachidonic acid, conversion of exogenously added arachidonic acid into MDA, or the action of prostaglandins; (d) both flurazepam and diazepam inhibit PLC-mediated activation of platelets; and (e) neither diazepam nor flurazepam achieve their antiplatelet actions by affecting platelet cyclic nucleotide levels.

Adenosine Diphosphate↗
PubMed1985

Antagonism of prostaglandin-mediated responses in platelets and vascular smooth muscle by 13-azaprostanoic acid analogs. Evidence for selective blockade of thromboxane A2 responses.

Studies were undertaken to examine the pharmacological properties and stereochemical requirements of a limited series of prostanoic acid analogs for inhibition of arachidonic acid (AA) and/or endoperoxide (U46619)-mediated responses in human platelets and rat aorta. To assess the role of stereochemistry, a set of trans- and cis-isomers of 13-azaprostanoic acid (APA) and 11a-homo-13-azaprostanoic acid (HAPA) were prepared. Each prostanoic acid analog blocked AA- or U46619-induced aggregatory and secretory responses in platelets, and U46619-mediated contractions of rat aorta in a concentration-dependent manner (0.1 to 100 microM). The azaprostanoic acid analogs blocked responses to both inducers of platelet activation with IC50 values ranging from 3.4 to 27.5 microM. Trans-APA was about 2- to 3-fold more active as an antagonist of serotonin release induced by AA or U46619 than the remaining analogs. The rank order of inhibitory potency (IC50; microM) for these analogs against U46619-induced serotonin release in human platelets was trans-APA (3.4) greater than cis-APA (8.9) = cis-HAPA (8.7) = trans-HAPA (9.1). Concentrations of the prostanoic acid analogs required to block these responses to AA and U46619 were similar, and the highest concentration used (100 microM) did not modify AA-induced malondialdehyde production in human platelet preparations. In contrast, the isomers of APA and HAPA were equally active as antagonists of U46619-induced contractions of rat vascular tissue, possessing KB values varying from 7.1 to 13.2 microM. Each azaprostanoic acid analog shifted the concentration-response curve of U46619 in rat aorta to the right, indicating a competitive-type inhibition. In addition, the azoprostanoic acid analog (U51605) was a more potent competitive antagonist of U46619 in this preparation and possessed an average pKB value of 6.18. In summary, the results show that (1) expansion of the five-membered ring of APA to the six-membered ring analogs (HAPA) led to a retention of potent inhibitory activity against U46619 in human platelets and rat vascular smooth muscle, (2) the antiaggregatory and antisecretory actions of the azaprostanoic acid analogs were mediated by a blockade of the responses to AA and U46619, and not by an inhibition of AA metabolism, (3) the blocking activity for the APA isomers was stereoselective (trans greater than cis) whereas the isomers of HAPA were equally effective as inhibitors of platelet function; and (4) these azaprostanoic acid analogs act as selective endoperoxide (U46619)/thromboxane A2 antagonists in these two tissues.

Adult↗
PubMed1984

Human platelet activation by bacterial phospholipase C is mediated by phosphatidylinositol hydrolysis but not generation of phosphatidic acid: inhibition by a selective inhibitor of phospholipase C.

We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes human platelet aggregation and secretion in a concentration dependent manner. The present study was undertaken to further characterize the specificity of the effects of PLC and to better understand the mechanism of the action of this inducer. A methylene-dioxybenzazepine (MDBA) analog of trimetoquinol was synthesized and tested for antiplatelet activity. MDBA (3-30 microM) inhibited PLC-induced aggregation in a concentration dependent manner. Whereas up to 200 microM MDBA did not inhibit aggregation induced by either thrombin, arachidonic acid, or U46619. Effects of PLC (0.05 U/ml) on hydrolysis of phosphatidylinositol, production of phosphatidic acid and thromboxane B2 (TXB2) synthesis were investigated using [32P]-phosphate and [14C]-arachidonic acid labeled platelets. PLC (0.05 U/ml) caused a time dependent decrease in platelet phosphatidylinositol. Up to 50% of labeled phosphatidylinositol was lost from platelets in five minutes. MDBA (3-30 microM) inhibited PLC-induced loss of phosphatidylinositol in a concentration dependent manner. An increase in phosphatidic acid was also observed in PLC-stimulated platelets. Up to 100 microM MDBA did not inhibit production of phosphatidic acid. PLC-treated platelets did not produce any TXB2. In other experiments possible protease contamination of PLC preparations was tested by incubating PLC (0.03-0.5 U/ml) with [14C]-casein. PLC in concentrations up to ten times higher than the concentrations used in aggregation studies did not cause hydrolysis of [14C]-casein, whereas more than 30% of [14C]-casein was hydrolyzed by trypsin. PLC-induced aggregation was not inhibited by up to 300 microM adenosine or ATP. In other experiments, platelet aggregation by ADP was inhibited by adenosine and ATP in a concentration dependent manner. The addition of calcium (0.5- 2.0 mM) increased aggregation by PLC in a concentration dependent manner. These findings suggest that PLC-induced activation of platelets is: (a) dependent on phosphatidylinositol hydrolysis but not on the production of phosphatidic acid, TXB2 or secretion of ADP; (b) not caused by protease contaminants; (c) calcium dependent; and (d) MDBA inhibits PLC-induced aggregation by blocking phosphatidylinositol hydrolysis.

Adenosine↗
PubMed1983

Acetylcholine receptor turnover in clonal muscle cells: role of plasmin and effects of protease inhibitors.

Characteristics of acetylcholine receptors were evaluated in G8-1, a continuous skeletal muscle line. Peak binding of 125I-alpha-bungarotoxin was in 10-day-old contractile myotubes at 4-8 nm. Turnover was studied using two different methods; both indicated half-times as little as half as long as previously reported for primary cultures. The effects of a variety of protease inhibitors on receptor turnover were assessed to determine if G8-1 receptors were less stable or turned over faster because of increased neutral protease activity. Leupeptin, antipain, and chloroquine markedly slowed receptor degradation. Inhibitors of plasmin or plasminogen activator had definite but less dramatic effects on receptor turnover. Results from studies in which plasmin was increased in the tissue culture media indicated that a small but definite acceleration of receptor turnover occurred. In clonal G8-1 cells, total number of acetylcholine receptors is controlled by negative feedback and although the major pathway for receptor degradation is lysosomal, plasmin may play a role in initiating receptor internalization.

Animals↗
PubMed1982

Plasminogen activator: the major secreted neutral protease of cultured skeletal muscle cells.

Clonal mouse skeletal muscle cells which differentiate in culture and form synapses with neuronal cells were found to secrete high levels of protease activity as measured with an 125I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean trypsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occurred with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromuscular contacts in experimental and pathologic denervation.

Animals↗