Search PubMed⌕ Search

Biomedical subjects

S M Seiler

Publications and source records attributed to S M Seiler.

33 records · Page 2Linked to original sources
PubMed1993

Binding and signal transduction of the cloned vascular angiotensin II (AT1a) receptor cDNA stably expressed in Chinese hamster ovary cells.

The vascular angiotensin (A) II receptor cDNA (AT1a) was transfected into Chinese hamster ovary (CHO) cells to generate the stable cell line CHO-AT1a. This cell line was used to investigate the binding and signal transduction properties of the cloned vascular AT1 receptor. Specific binding of sarcosine1(-)[125I]tyrosine4-isoleucine8-AII ([125I]SI-AII) to CHO-AT1a membranes reached equilibrium after 1 h at 25 degrees C and was consistently greater than 95% of total binding. Saturation binding analyses demonstrated [125I]SI-AII bound to a saturable population of sites on membranes with an equilibrium dissociation constant (KD) of 0.7 nM and a binding site maximum of 1.2 pmol/mg protein. [125I]SI-AII binding to CHO cells was inhibited by the following compounds with a rank order of potency of SI-AII > AII > losartan > AI >> PD 123,177. AII (1 microM) treatment of CHO-AT1a cells caused an increase in inositol phosphates and intracellular calcium relative to basal levels. These responses were blocked by losartan but not by PD 123,177. AII (1 microM) did not effect adenylate cyclase activity in CHO-AT1a cells, whereas the agonist inhibited adenylate cyclase activity in rat liver cell membranes. These effects were blocked by 10 microM losartan. These results indicate that CHO-AT1a cells express functional AT1a receptors which stimulate phospholipase C activity but not adenylate cyclase activity. CHO-AT1a cells should provide a useful model for studies of AT1a receptor domains which are critical to signaling pathways.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1993

Prostacyclin agonists reduce early atherosclerosis in hyperlipidemic hamsters. Octimibate and BMY 42393 suppress monocyte chemotaxis, macrophage cholesteryl ester accumulation, scavenger receptor activity, and tumor necrosis factor production.

We determined the effects of two prostacyclin agonists (octimibate and BMY 42393) on the progression of the fatty streak in vivo and on macrophage function in vitro. Hamsters were fed chow plus 0.05% cholesterol and 10% coconut oil. Control hamsters were compared with animals receiving either octimibate (10 or 30 mg/kg per day) or BMY 42393 (30 mg/kg per day). After 10 weeks of treatment, octimibate decreased plasma total cholesterol and triglycerides by 43% and 32%, respectively. Neither agonist affected blood pressure or heart rate. Lesion-prone aortic arches were stained with hematoxylin and oil red O and examined en face. Compared with controls, octimibate and BMY 42393 on average decreased mononuclear cells attached to the luminal surface by 44% and reduced subendothelial macrophage-foam cell number by 56%, foam cell size by 38%, and fatty streak area by 63%. Since octimibate is a putative inhibitor of acyl coenzyme A cholesterol acyltransferase, we studied the effect of both agents on cholesteryl ester metabolism in murine macrophages. At 10 microM, octimibate and BMY 42393 decreased cholesteryl ester accumulation in macrophages by 90% and 41%, respectively. Octimibate inhibited cholesteryl ester synthesis by 96% and increased the rate of cholesteryl ester degradation by 52%. Both prostacyclin agonists reduced macrophage scavenger receptor-mediated uptake of acetylated low density lipoprotein by 24-66% and increased cyclic adenosine monophosphate levels. Octimibate and BMY 42393 inhibited the secretion of tumor necrosis factor by 80-88% when macrophages were activated with lipopolysaccharide. At 10 microM, both agents decreased human monocyte chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by 64-79%. The in vitro results with octimibate and BMY 42393 are consistent with the low number of small foam cells quantified in vivo. We suggest that octimibate and BMY 42393 suppress monocyte-macrophage atherogenic activity and cytokine production and thus inhibit the development of early atherosclerosis.

Animals↗
PubMed1993

Comparison of thrombin active site and exosite inhibitors and heparin in experimental models of arterial and venous thrombosis and bleeding.

Different pharmacological approaches to thrombin inhibition were compared for their effects on thrombosis and bleeding time in anesthetized rats. Thrombosis was induced in the carotid artery by transmural vessel injury and in the vena cava by partial blood flow stasis combined with mild endothelial disruption. Small mesenteric arteries were punctured with a hypodermic needle to measure the bleeding time. Dose-response relationships were determined with a thrombin active site inhibitor, N-methyl (GYKI 14,766); a thrombin exosite inhibitor, succinyl-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-Gln (BMS 180,742); and heparin. BMS 180,742 interferes with fibrinogen binding to the thrombin exosite but, unlike GYKI 14,766, it does not block thrombin's catalytic site. The effects on thrombosis and bleeding time were correlated with ex vivo clotting times using the activated partial thromboplastin time for heparin and the thrombin time for GYKI 14,766 and BMS 180,742. Venous thrombosis was inhibited more than 90% by all three inhibitors at doses that either produced threshold increases or had no effect on bleeding and clotting times. Arterial thrombosis was inhibited 82% by GYKI 14,766 and 63% by heparin but it was not inhibited by BMS 180,742. These antithrombotic activities were accompanied by a maximal activated partial thromboplastin time increase and doubling of the bleeding time with heparin and a maximal thrombin time prolongation and 35% increase in bleeding time with GYKI 14,766. These results suggest that thrombin inhibitors, which act at the active site or exosite or through antithrombin III, are equally efficacious against venous thrombosis but active site inhibitors are the most effective against arterial thrombosis.

Amino Acid Sequence↗
PubMed1992

Involvement of the "tethered-ligand" receptor in thrombin inhibition of platelet adenylate cyclase.

Thrombin is thought to activate platelets through multiple signaling pathways. Recently a new thrombin receptor was identified (Vu et al., Cell 64:1057-1068, 1991) that recognizes alpha-thrombin's anion-binding exosite. Thrombin cleaves this receptor generating a new N-terminal ("tethered-ligand") that activates the receptor. We report here that this receptor is involved in alpha-thrombin inhibition of platelet adenylate cyclase, a process thought mediated by thrombin's high-affinity pathway. In gel-filtered human platelets, iloprost-stimulated cAMP levels were lowered by alpha- and zeta-thrombin addition and, to a much lesser extent, by gamma-thrombin. The alpha- and zeta-thrombin mediated decreases in cAMP were prevented by the thrombin anion-binding exosite inhibitor, BMS 180742, implying that binding to thrombin's anion-binding exosite was required. The iloprost-stimulated increase in cAMP was also reversed (in a concentration-dependent fashion) by a peptide mimicking the new N-terminal of the "tethered-ligand" thrombin receptor (SFLLRNPNDKYEPF). In broken cell preparations, platelet adenylate cyclase activity was also inhibited by SFLLRNPNDKYEPF (but not by a similar peptide used as a control, FSLLRNPNDKYEPF). These results support the hypothesis that thrombin inhibition of platelet adenylate cyclase activity is mediated, at least in part, via the "tethered-ligand" receptor. Moreover, this data is consistent with the "tethered-ligand" receptor mediating the high affinity actions of alpha-thrombin.

Adenylyl Cyclase Inhibitors↗
PubMed1992

Structure-activity relationships associated with 3,4,5-triphenyl-1H-pyrazole-1-nonanoic acid, a nonprostanoid prostacyclin mimetic.

A series of phenylated pyrazoloalkanoic acid derivatives were synthesized and evaluated as inhibitors of ADP-induced human platelet aggregation. 3,4,5-Triphenyl-1H-pyrazole-1-nonanoic acid (8d), with an IC50 of 0.4 microM, was the most potent inhibitor identified in this study. Biochemical studies determined that 8d increased intraplatelet cAMP accumulation and stimulated platelet membrane-bound adenylate cyclase in a concentration-dependent fashion. Displacement of [3H]iloprost by 8d from platelet membranes indicated that the platelet prostacyclin (PGI2) receptor is the locus of biological action. Structure-activity studies demonstrated that the minimum structural requirements for binding to the platelet PGI2 receptor and inhibition of ADP-induced platelet aggregation within this series are a vicinally diphenylated pyrazole substituted with an omega-alkanoic acid side chain eight or nine atoms long. Potency depended upon both side-chain length and its topological relationship with the two phenyl rings.

Adenylyl Cyclases↗
PubMed1991

Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor.

Recently a thrombin receptor with a unique mechanism of activation was cloned from a megakaryocyte-like cell line (Vu et al., Cell 64:1057-1068, 1991). Thrombin cleaves a portion of this receptor creating a new N-terminus that acts as a "tethered-ligand" to activate the receptor. A thrombin receptor activating peptide (SFLLRNPNDKYEPF) homologous to the new N-terminus was shown to activate platelets. We synthesized this peptide and demonstrated that it desensitized platelets to activation by low concentrations of alpha-thrombin but not gamma-thrombin. We also synthesized a thrombin exosite inhibitor (BMS 180742) that inhibited platelet aggregation induced by low, but not high, concentrations of alpha-thrombin. In contrast, a thrombin active site inhibitor, N alpha-(2-naphthylsulfonyl-glycyl)-D,L-amidinophenylalanylpiperi dide, competitively inhibited thrombin-induced platelet aggregation. We conclude that thrombin-induced platelet activation is mediated by at least two pathways: one activated by low concentrations of alpha-thrombin and blocked by a thrombin exosite inhibitor that appears to be coupled to the "tethered-ligand" thrombin receptor, and another that is stimulated by higher concentrations of alpha-thrombin and by gamma-thrombin and does not require the thrombin exosite for activation. Both pathways are blocked by a thrombin active site inhibitor.

Amino Acid Sequence↗
PubMed1991

Antithrombotic activity of BMY-43351, a new imidazoquinoline with enhanced aqueous solubility.

BMY-43351 is a new broad-spectrum inhibitor of platelet aggregation with greater aqueous solubility than earlier analogs from the imidazoquinoline series. This report compares the antithrombotic activity of BMY-43351 to that of two other imidazoquinolines: BMY-20844, a simply-substituted compound, and BMY-21638, a more potent ether-linked side chain analog. All of these compounds act, at least in part, via inhibition of platelet low-Km cyclic AMP phosphodiesterase. Antithrombotic activity was assessed in the rabbit ear chamber-biolaser preparation, an animal model of small vessel thrombosis, and in the canine coronary artery stenosis-occlusion model of large vessel thrombosis. BMY-43351 was found to be remarkably potent in the biolaser model, with an EDso of 0.074 mg/kg p.o. In comparison, compounds such as aspirin, ticlopidine, sulfinpyrazone, and dipyridamole demonstrate little or no activity at much higher doses, (eg. 100 mg/kg p.o.). Other inhibitors of platelet low Km cyclic AMP phosphodiesterase are active but substantially weaker than BMY-43351. Similarly, in the coronary artery stenosis-occlusion model, BMY-43351 demonstrated impressive activity, significantly inhibiting arterial thrombosis at intraduodenal doses as low as 1 micrograms/kg. The potential use of BMY-43351 as adjunct therapy in thrombolysis was suggested in a series of experiments where this drug was used in combination with a thrombolytic regimen of stretokinase plus heparin. In this experimental setting, time to reperfusion was reduced from 42 +/- 5 minutes to 11 +/- 5 minutes, and reocclusion was totally inhibited.

Animals↗
PubMed1990

Relation of silent myocardial ischemia after coronary artery bypass grafting to angiographic completeness of revascularization and long-term prognosis.

The prevalence and characteristics of silent myocardial ischemia as detected by 24-hour ambulatory electrocardiography ST-segment depression were prospectively assessed in 94 patients examined early (1 to 3 months) and 184 patients examined late (12 months) after coronary artery bypass grafting (CABG), and followed for a mean of 48 +/- 11 (range 4 to 62) months. The relation of ambulatory electrocardiographic silent ischemia to evidence of completeness of revascularization as defined by cardiac angiography performed 1 and 12 months after CABG, and to prognosis by follow-up of adverse clinical events was analyzed. Silent ischemia was detected early in 20% (19 of 94) and late in 27% (50 of 184) of patients, and showed a mean frequency of episodes ranging from 6 to 10 episodes/24 hours with a mean duration ranging from 15 to 23 minutes. The circadian distribution of episodes disclosed a significant peak of ischemic activity during the period of 6 A.M. to noon and a secondary peak between 6 P.M. and midnight (p less than 0.01 and p less than 0.001, respectively). Silent ischemia was not found by univariate analysis to be associated with graft or anastomotic site occlusions, low graft flow rates, grafted arteries with significant distal residual stenoses or ungrafted stenotic native coronary arteries. Kaplan-Meier analysis of time to cardiac event showed that silent ischemia was not predictive of an adverse clinical event in the early years after CABG. Cox regression analysis of 30 covariates only disclosed age (relative risk 1.06 [95% confidence interval, 1.01 to 2.94]) as having an effect on time to adverse clinical event.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiography↗
PubMed1990

Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Effects on the Ca2+ release channel.

The Ca2+ release channel and ryanodine receptor are activities copurifying with the 400,000-450,000 Da high molecular weight protein of cardiac and skeletal junctional sarcoplasmic reticulum. Calpain II, an endogenous cytosolic protease, was used to selectively degrade the high molecular weight protein in cardiac and skeletal muscle sarcoplasmic reticulum vesicles, and its effects on the activity of the Ca2+ release channel and [3H]ryanodine binding sites were analyzed. Degradation of the high molecular weight protein was associated with appearance of 315,000 and 150,000 Da proteolytic fragments and with a change in the ultrastructure of the "feet," extravesicular projections that protrude from the junctional sarcoplasmic reticulum membrane. The maximal number of [3H]ryanodine binding sites and the affinities of the sites for ryanodine were not remarkably affected by calpain II. Ca2+ release channels recorded from nondegraded cardiac and skeletal membrane vesicle preparations had slope conductances of 85 and 110 pS, respectively, measured with 1 microM cis-Ca2+ and 50 mM trans-Ba2+. Proteolysis did not alter the unitary channel conductances but did increase the percentage of channel open times from 36% to more than 90%. After proteolysis, channel opening remained dependent on micromolar cis-Ca2+, and high concentrations of ryanodine (300 microM) still blocked the channel. Our results suggest that proteolysis of the Ca2+ release channel with calpain II selectively impairs its inactivation, leaving its unitary conductance and the requirement for micromolar Ca2+ intact.

Animals↗
PubMed1989

Natural history of potentially lethal ventricular arrhythmias in patients treated with long-term antiarrhythmic drug therapy.

To examine the natural history of long-term anti-arrhythmic therapy in patients with benign and potentially lethal ventricular premature complexes (VPCs), 28 patients with initial efficacy with moricizine (greater than 75% suppression of baseline mean VPCs/hr and greater than 90% suppression of repetitive VPCs) were prospectively followed for 1 to 56 (mean +/- standard deviation 25 +/- 17) months. Patients were examined during baseline placebo, anti-arrhythmic drug therapy and intermittent pulsed-placebo reexamination periods. The mean VPCs of all patients at baseline entry were 233 +/- 47 VPCs/hr, and after moricizine therapy 14 +/- 4 VPCs/hr. Follow-up demonstrated that antiarrhythmic efficacy decreased to 75% at 12 months and to 62% at 24 months. Loss of antiarrhythmic drug efficacy most commonly occurred as a "transient" event (10 patients [36%]), and efficacy was spontaneously reestablished without a change in antiarrhythmic therapy. In contrast, increased dose titration of moricizine was necessary to reestablish antiarrhythmic suppression efficacy in 4 patients (14%), and 4 patients (14%) lost antiarrhythmic drug responsiveness during follow-up. Spontaneous decrease in baseline VPCs resulted in discontinuation of antiarrhythmic therapy in 3 patients, and increase in baseline VPCs was associated with a loss of antiarrhythmic response in 2 patients. Late proarrhythmic effects (2 patients, 7%), delayed side effects necessitating drug withdrawal (6 patients, 21%) and medical events (4 patients, 14%) occurred during 56 months of follow-up. Individual serum moricizine levels remained in the therapeutic range throughout the study and did not correlate with changes in antiarrhythmic efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-Arrhythmia Agents↗
PubMed1989

Pharmacology of a potent, new antithrombotic agent, 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844).

The effects of 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844) on platelet function and experimental thrombosis were evaluated in a series of in vitro, ex vivo and in vivo experiments. The compound inhibited platelet aggregation in vitro in platelet rich plasma obtained from humans, rats and rabbits with EC50s of less than 1 microgram/ml when aggregation was induced by ADP, collagen or thrombin. Supra-additive interaction against ADP aggregation was also observed when BMY-20844 was combined with prostacyclin. BMY-20844 was orally active with an ex vivo ED50 in the rat of 3.2 mg/kg vs ADP. Significant antithrombotic activity was observed in two animal models (laser induced thrombosis in the microcirculation of the rabbit ear and coronary artery thrombosis in the dog). Inhibitions of 52% at 3 mg/kg p.o. in the laser model and 100% at 1 mg/kg i.d. in the coronary artery thrombosis model were obtained. Modest inotropic and hemodynamic effects were observed in ferrets and dogs. BMY-20844 was found to be a potent, specific inhibitor of platelet low Km cyclic AMP phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels.

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.

Adenosine Triphosphate↗
PubMed1987

Inhibitors of inositol trisphosphate-induced Ca2+ release from isolated platelet membrane vesicles.

Platelet membrane vesicles accumulated Ca2+ in an ATP-dependent fashion, and 25-50% of the accumulated Ca2+ was released by the addition of 10 microM inositol 1,4,5-trisphosphate (IP3). The concentration of IP3 required for half-maximal Ca2+ release was approximately 0.5 microM. The inhibition of IP3-induced Ca2+ release from these membrane vesicles by various agents was examined. Of the plasma membrane Ca2+ channel blockers, cinnarizine and flunarizine were found to be potent inhibitors of IP3-induced Ca2+ release while having no effect on ATP-dependent Ca2+ uptake. The IC50 value for both cinnarizine and flunarizine as inhibitors of IP3-induced Ca2+ release was below 10(-6) M. Nifedipine, verapamil, bepridil, and diltiazem did not significantly inhibit IP3-induced Ca2+ release at the highest concentration tested (50 microM). The "intracellular Ca2+ antagonists" ryanodine, TMB-8 (8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate), dantroline, trifluoperazine and chlorpromazine were not inhibitors of IP3-induced Ca2+ release at 50 microM. The local anesthetics benzocaine and lidocaine weakly inhibited the IP3-induced Ca2+ release with IC50 values of approximately 5 and 50 microM, respectively, whereas other local anesthetics tested were less potent inhibitors. The potent inhibitors described may prove useful as probes of the IP3-induced Ca2+ release channels.

Adenosine Triphosphate↗
PubMed1985

Demonstration of a Na+/H+ exchange activity in purified canine cardiac sarcolemmal vesicles.

Purified canine cardiac sarcolemmal membrane vesicles exhibit a sodium ion for proton exchange activity (Na+/H+ exchange). Na+/H+ exchange was demonstrated both by measuring rapid 22Na uptake into sarcolemmal vesicles in response to a transmembrane H+ gradient and by following H+ transport in response to a transmembrane Na+ gradient with use of the probe acridine orange. Maximal 22Na uptake into the sarcolemmal vesicles (with starting intravesicular pH = 6 and extravesicular pH = 8) was approximately 20 nmol/mg protein. The extravesicular Km of the Na+/H+ exchange activity for Na+ was determined to be between 2 and 4 mM (intravesicular pH = 5.9, extravesicular pH = 7.9), as assessed by measuring the concentration dependence of the 22Na uptake rate and the ability of extravesicular Na+ to collapse an imposed H+ gradient. All results suggested that Na+/H+ exchange was reversible and tightly coupled. The Na+/H+ exchange activity was assayed in membrane subfractions and found most concentrated in highly purified cardiac sarcolemmal vesicles and was absent from free and junctional sarcoplasmic reticulum vesicles. 22Na uptake into sarcolemmal vesicles mediated by Na+/H+ exchange was dependent on extravesicular pH, having an optimum around pH 9 (initial internal pH = 6). Although the Na+/H+ exchange activity was not inhibited by tetrodotoxin or digitoxin, it was inhibited by quinidine, quinacrine, amiloride, and several amiloride derivatives. The relative potencies of the various inhibitors tested were found to be: quinacrine greater than quinidine = ethylisopropylamiloride greater than methylisopropylamiloride greater than dimethylamiloride greater than amiloride. The Na+/H+ exchange activity identified in purified cardiac sarcolemmal vesicles appears to be qualitatively similar to Na+/H+ exchange activities recently described in intact cell systems. Isolated cardiac sarcolemmal vesicles should prove a useful model system for the study of Na+/H+ exchange regulation in myocardial tissue.

Alamethicin↗