Hypertriglyceridemic VLDL decreases plasminogen binding to endothelial cells and surface-localized fibrinolysis.

The effect of normo (NTG)- and hypertriglyceridemic (HTG)-VLDL on cultured human umbilical vein endothelial cell (HUVEC) surface-localized fibrinolysis was examined following pre-incubation with NTG-, HTG-VLDL, LDL (1-20 micrograms/mL) or buffer (control). Ligand binding assays, using 125I-labeled tcu-PA, t-PA, or Glu-plasminogen (Glu-Pmg) were carried out in the absence/presence of lipoproteins. Scatchard analyses showed that HTG-VLDL decreased the Bmax for 125I-labeled Glu-Pmg ligand binding approximately 35% [(2.11 +/- 0.39)-(1.40 +/- 0.32) x 10(6) sites/cell, p < 0.005] and increased the Kd, app approximately 5-fold (0.32 +/- 0.03 to 1.74 +/- 0.08 microM, p < 0.01), while NTG-VLDL, LDL, and buffer had no effect. 125I-labeled PA ligand binding was unaffected by these lipoproteins. Receptor-bound PA activation of cell-bound 125I-labeled Glu-Pmg was measured by quantitation of either the M(r) 20 kDa light- or M(r) 60 kDa heavy-chain of 125I-labeled plasmin, following SDS-PAGE. Kinetic analysis of these data (HTG-VLDL vs controls) indicated that HTG-VLDL decreased the V(max) of tcu-PA- and t-PA-mediated activation of plasminogen approximately 2.7-fold (0.317 +/- 0.023 vs 0.869 +/- 0.068 nM s-1, p < 0.01) and approximately 2.9-fold (0.391 +/- 0.098 vs 1.152 +/- 0.265 nM s-1, p < 0.01), respectively. Increasing concentrations of the HTG-VLDL increased 1/V(max), yielding a series of parallel plots, typical for uncompetitive inhibition with a Ki for inhibition of approximately 10 micrograms/mL. The combined ligand binding and kinetic data best fit an uncompetitive inhibition model in which the binding of the large HTG-VLDL particle to the EC surface may directly affect Glu-Pmg binding and activation, thus contributing to early fibrin deposition and the increased thrombotic risk associated with HTG.

Thrombin decreases the urokinase receptor and surface-localized fibrinolysis in cultured endothelial cells.

The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (< 5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluoro-phosphate-tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase-polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 6-fold more 35-kD M(r) 35S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and the glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) were also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.

Metabolism and disposition of the dopaminergic agonist 5-hydroxy-6-methyl-2-di-n-propylaminotetralin in the rat.

As part of a program to investigate the metabolism and disposition of putative dopamine receptor agonists, DK-118 (5-hydroxy-6-methyl-2-di-n-propylaminotetralin) was chosen for study in the rat. Following a 3.85 mg/kg (ip) dose of 5-hydroxy[6-14C]methyl-2-di-n-propylaminotetralin, an average (+/- SD) of 100.3 +/- 12.2% was recovered in 67 hr: 77.2 +/- 7.9% in urine and 23.1 +/- 6.2% in feces. No excretion of 14CO2 was observed. In bile duct-cannulated animals, an average of 31.6% of the dose was recovered in the bile within 6 hr. After injection of bile containing radiolabeled drug/metabolites into the lumen of the duodenum, 30.2 +/- 1.7% of the injected radioactivity was recovered in the urine, suggesting enterohepatic circulation of some of the drug/metabolites excreted in bile. Highest concentrations of tissue radioactivity, 0.5 hr after ip injection of 14C-DK-118, were found in lung, kidney, and liver. Only a small amount of unchanged DK-118 is excreted into urine and bile; HPLC radiochromatography separated five metabolites in urine and at least eight metabolites in bile. The three major metabolites in urine (70% of urinary radioactivity) have been identified as 5-hydroxy-6-carboxy-2-di-n-propylaminotetralin, 5-hydroxy-6-carboxy-2-n-propylaminotetralin, and 5-hydroxy-6-methyl-2-n-propylaminotetralin-O-sulfate. The two major biliary metabolites have been identified as 5-hydroxy-6-carboxy-2-n-propylaminotetralin and an acid-labile conjugate of DK-118. Together, these data indicate that DK-118 is metabolized in the rat by a combination of N-dealkylation, oxidation of the 6-methyl carbon, and conjugation with sulfate.

Effects of metyrapone on the pharmacological activity, plasma levels and urinary excretion of the dopamine receptor agonist DK-118.

Possible metabolic activation of the dopamine receptor agonist DK-118 (5-hydroxy-6-methyl-N,N-di-n-propyl-2-aminotetralin) was investigated in cats. Metyrapone, an inhibitor of oxidative drug metabolism, was given to cats before DK-118 and the pharmacologic effects of the dopamine agonist were compared to those observed in nonpretreated animals. A sensitive high-performance liquid chromatography assay using electrochemical detection was developed to monitor urine and plasma concentrations of DK-118 in metyrapone-pretreated and control animals. The DK-118-mediated inhibition of cardioaccelerator nerve stimulation-induced tachycardia was reduced markedly in cats pretreated with metyrapone but the pretreatment had no effect on the hypotension or bradycardia produced by DK-118. In a separate group of cats, the tachycardia inhibitory effect of a nonbioactivated dopamine agonist, dipropyldopamine, was unaffected by metyrapone pretreatment, confirming that the inhibitor of drug metabolism does not interfere with this dopamine receptor-mediated effect. Pretreatment with metyrapone before a 0.14-mumol/kg i.v. dose of DK-118 increased the half-life, reduced total drug clearance and increased urinary excretion of unchanged DK-118. All of the changes are consistent with a metyrapone-related inhibition of DK-118 metabolism. The results of this study show that inhibition of DK-118 metabolism reduces certain of its pharmacologic actions, indicating that one or more of the metabolites of the drug may contribute to its effects.

In vivo and in vitro dopaminergic effects of three ergoline fragments.

The pharmacological effects of three ergoline fragments (BD-179, BD-271 and BD-214) were studied in vivo using the cat cardioaccelerator nerve preparation and in vitro using field stimulated isolated cat right atria. BD-179 and BD-271 produced dose dependent inhibition of tachycardia due to electrical stimulation of the right postganglionic cardioaccelerator nerve in anesthetized cats, BD-214 was inactive. BD-179 produced primarily hypotension, BD-271 produced a transient pressor response followed by hypotension and BD-214 produced only pressor effects. The tachycardia inhibitory effects and hypotension produced by BD-179 and BD-271 were antagonized by the dopamine receptor antagonist sulpiride. BD-179, BD-271 and BD-214 antagonized the presynaptic inhibitory effects of the dopamine receptor agonist apomorphine in vitro on field stimulated isolated cat right atria. All three ergoline fragments facilitated stimulation-induced increases in systolic tension development and BD-214 facilitated stimulation-induced tachycardia in isolated cat right atria.

Possible biologically active metabolites of 5-hydroxy-6-methyl-2-di-n-propylaminotetralin.

5-Hydroxy-6-methyl-2-di-n- propylaminotetralin 1b exhibits prominent peripheral presynaptic and central dopaminergic effects, and pharmacological test data suggest that this compound is metabolically activated in vivo. It was speculated that the 6-methyl group is oxidized. To evaluate this possibility and as a prelude to a metabolism study, 5-hydroxy-6-formyl 5 and 5-hydroxy-6-hydroxymethyl-2-di-n- propylaminotetralin 2b were synthesized. Both compounds exhibited marked potency/activity in a cat cardioaccelerator nerve preparation. The biological data on these compounds are consistent with the possibility of their being pharmacologically active metabolites of compound 1b.

Antagonist properties of phentolamine at both presynaptic alpha 2-adrenoceptors and presynaptic dopamine receptors using field stimulated right cat atria.

Phentolamine, which is considered a specific alpha-adrenoceptor antagonist, was tested for antagonist properties at presynaptic dopamine receptors and presynaptic alpha 2-adrenoceptors present on sympathetic nerve terminals in isolated right cat atria. Field stimulation induced a transient tachycardia which was inhibited by stimulation of presynaptic dopamine receptors using apomorphine or by stimulation of presynaptic alpha 2-adrenoceptors using clonidine. The presynaptic inhibitory effects of apomorphine were competitively antagonized by sulpiride, which is considered a specific dopamine receptor antagonist, and by phentolamine and yohimbine which are considered alpha-adrenoceptor antagonists. The presynaptic inhibitory effects of clonidine were competitively antagonized by phentolamine and yohimbine but not by sulpiride. pA2 values for phentolamine against apomorphine and phentolamine against clonidine suggest that phentolamine may be an antagonist at both presynaptic dopamine receptors and presynaptic alpha 2-adrenoceptors.