Peroxisome proliferator-activated receptor subtype-specific regulation of hepatic and peripheral gene expression in the Zucker diabetic fatty rat.

Fibrates and thiazolidinediones are used clinically to treat hypertriglyceridemia and hyperglycemia, respectively. Fibrates bind to the peroxisome proliferator-activated receptor (PPAR)-alpha, and thiazolidinediones are ligands of PPAR-gamma. These intracellular receptors form heterodimers with retinoid X receptor to modulate gene transcription. To elucidate the target genes regulated by these compounds, we treated Zucker diabetic fatty rats (ZDF) for 15 days with a PPAR-alpha-specific compound, fenofibrate, a PPAR-gamma-specific ligand, rosiglitazone, and a PPAR-alpha/-gamma coagonist, GW2331, and measured the levels of several messenger RNAs (mRNAs) in liver by real-time polymerase chain reaction. All 3 compounds decreased serum glucose and triglyceride levels. Fenofibrate and GW2331 induced expression of acyl-coenzyme A (CoA) oxidase and enoyl-CoA hydratase and reduced apolipoprotein C-III and phosphoenolpyruvate carboxykinase mRNAs. Rosiglitazone modestly increased apolipoprotein C-III mRNA and had no effect on expression of the other 2 genes in the liver but increased the expression of glucose transporter 4 and phosphoenolpyruvate carboxykinase in adipose tissue. We identified a novel target in liver, mitogen-activated phosphokinase phosphatase 1, whose down-regulation by PPAR-alpha agonists may improve insulin sensitivity in that tissue by prolonging insulin responses. The results of these studies suggest that activation of PPAR-alpha as well as PPAR-gamma in therapy for type 2 diabetes will enhance glucose and triglyceride control by combining actions in hepatic and peripheral tissues.

Design and synthesis of 2-methyl-2-[4-(2-[5-methyl-2-aryloxazol-4-yl]ethoxy)phenoxy]propionic acids: a new class of dual PPARalpha/gamma agonists.

Propionic acid derivative 8, which was designed and synthesized based on putative pharmacophores of known PPARgamma- and PPARalpha-selective compounds, exhibits potent dual PPARalpha/gamma agonist activity as demonstrated by in vitro binding and dose overlap in the newly introduced EOB mouse model for glucose lowering and lipid/cholesterol homeostasis.

Raloxifene and estrogen reduces progression of advanced atherosclerosis--a study in ovariectomized, cholesterol-fed rabbits.

The present study investigated the effect of raloxifene, a selective estrogen receptor modulator (SERM), on aortic atherosclerosis in 80 ovariectomized, cholesterol-fed rabbits with pre-induced atherosclerosis. The animals were fed an atherogenic diet containing 240 mg cholesterol/day for 15 weeks, after this period a baseline control group was sacrificed. Thereafter, oral treatment was initiated with either estradiol 4 mg/day (n=20), raloxifene (210 mg/day) or placebo (n=20). In the treatment period of 39 weeks, the dietary cholesterol content was reduced to 80 mg cholesterol/day. Postmortem evaluation showed a significantly increased uterine weight induced by estradiol treatment (10.3+/-1.2 g), whereas raloxifene intervention caused a decreased uterus weight (1.21+/-0.1 g) when compared to placebo (2.48+/-0.47 g). Throughout the study, serum lipids increased in all groups to levels seen in very high risk humans. After 58 weeks the cholesterol content in the aorta was 3.18+/-0.54 micromol/cm(2) (38% reduction) in the estradiol group, 3.66+/-0.52 micromol/cm(2) (29% reduction) in the raloxifene group and 5.12+/-0.60 micromol/cm(2) in the placebo group. Analyses of the aortic cholesterol content corrected for time-averaged serum cholesterol revealed that both estradiol and raloxifene therapy significantly reduced the progression of atherosclerosis (P<0.01 for both) as compared to placebo.

Raloxifene and estrogen inhibit neointimal thickening after balloon injury in the carotid artery of male and ovariectomized female rats.

The effects of raloxifene and 17alpha-ethinyl estradiol (EE2) on intimal thickening in response to balloon injury were tested in male and ovariectomized female rats. In male rats, oral raloxifene and EE2, administered either by gavage or in the diet, inhibited arterial intimal thickening in response to balloon injury to a maximum of approximately 60 and 50%, respectively. The effect of oral raloxifene to decrease cholesterol was observed at doses (> or = 3 mg/kg/day) higher than those required to inhibit intimal thickening (> or = 0.03 mg/kg/day). Coadministration of the estrogen receptor antagonist, ICI 182,780 (5 mg/kg/day, s.c.), blocked the inhibition of balloon injury by raloxifene and EE2. Direct adventitial delivery of raloxifene (0.03 mg/kg/day) and EE2 (0.001 mg/kg/day) to the vascular wall inhibited intimal thickening by 63 and 53%, respectively. In ovariectomized female rats, oral raloxifene (0.01-3.0 mg/kg/day) and EE2 (0.08 mg/kg/day) inhibited intimal thickening to a maximum of 32 and 60%, respectively. Together, these data suggest that raloxifene and EE2, inhibit balloon arterial injury in the rat through direct effects on the vascular wall that involve the estrogen receptor and are at least partially independent of serum cholesterol.

Raloxifene reduces atherosclerosis: studies of optimized raloxifene doses in ovariectomized, cholesterol-fed rabbits.

OBJECTIVE: We have previously shown that raloxifene, a selective oestrogen receptor modulator, 35 mg/day inhibits atherosclerosis in ovariectomized, cholesterol-fed rabbits. This effect was only partial as compared to 17beta-oestradiol 4 mg/day; however, plasma raloxifene concentrations were low relative to those obtained in raloxifene-treated women. We therefore investigate the effects of raloxifene at higher doses. DESIGN: The study on atherosclerosis in ovariectomized, cholesterol-fed rabbits (n = 80) compared raloxifene 70 mg/day and 210 mg/day to 17beta-oestradiol 4 mg/day and placebo. RESULTS: After 48 weeks of therapy, the aortic cholesterol content in the 70 mg/day and 210 mg/day raloxifene treatment groups were 471 +/- 56 nmol/mg protein and 456 +/- 56 nmol/mg protein, respectively. This was significantly less than in the placebo group (654 +/- 69 nmol/mg protein; P < 0.05). In the oestrogen-treated group, the aortic cholesterol content was 357 +/- 62 nmol/mg protein (P < 0.01 as compared to placebo). Differences in serum lipids between the treatment groups could only partly explain the effect on aortic cholesterol content, indicating that additional anti-atherogenic mechanisms may contribute to the decrease in aortic atherosclerosis. This anti-atherosclerotic activity of raloxifene was observed at plasma concentrations comparable to those in postmenopausal women during raloxifene treatment. CONCLUSIONS: We conclude that clinically relevant raloxifene treatment inhibits aortic atherosclerosis in ovariectomized, cholesterol-fed rabbits.

Effects of LY295427, a low-density lipoprotein (LDL) receptor up-regulator, on LDL receptor gene transcription and cholesterol metabolism in normal and hypercholesterolemic hamsters.

The action of LY295427 [(3alpha,4alpha, 5alpha)-4-(2-propenylcholestan-3-ol)], a compound that derepresses low-density lipoprotein receptor (LDL-R) expression in a cell-based model, was examined in hamsters. It was found that the compound does not have an effect in normal chow-fed hamsters, in which LDL-R levels are not repressed, but exerts a marked hypocholesterolemic effect (>70% decrease) in cholesterol-coconut oil-fed hamsters, in which LDL-R is repressed. In this model, there is a dose-response for cholesterol lowering with an approximate ED50 value of 40 mg/kg/day and an inverse relationship between serum cholesterol and serum LY295427 levels. LDL-R mRNA is increased (2-fold) and liver cholesterol ester content is decreased (>90%). Unlike the 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor lovastatin, the decreased serum cholesterol is confined to the non-high-density lipoprotein fraction. Furthermore, LY295427 does not affect cholesterol biosynthesis, and it does not have a significant effect on cholesterol absorption. These data suggest that LY295427 acts in the hypercholesterolemic hamster by derepressing LDL-R transcription, thereby enhancing cholesterol clearance from the blood. The results with LY295427 suggest that compounds that act to increase LDL-R may represent a novel approach in the pharmacotherapy for hypercholesterolemia.

Raloxifene inhibits aortic accumulation of cholesterol in ovariectomized, cholesterol-fed rabbits.

BACKGROUND: The beneficial effect of long-term hormone replacement therapy in terms of a decreased risk of cardiovascular disease is now generally accepted. Raloxifene, a selective estrogen receptor modulator, has demonstrated hypolipidemic properties while leaving the endometrium unstimulated. METHODS AND RESULTS: For our study of the effects of raloxifene on atherosclerosis, 75 rabbits were ovariectomized and treated with either raloxifene, 17beta-estradiol, or placebo; 25 rabbits were sham operated and treated with placebo. After 45 weeks, the raloxifene group had two thirds of the aortic atherosclerosis, as evaluated by the cholesterol content of the proximal inner part of the aorta, found in the placebo group (placebo, 577+/-55.1 nmol/mg protein; raloxifene, 397+/-53.6 nmol/mg protein; P<.05); the estrogen group had one third of the aortic atherosclerosis in the placebo group (estrogen, 177+/-32.1 nmol/mg protein; P<.001). The sham-operated group (473+/-59.6 nmol/mg protein) was not significantly different from placebo. These effects were only partly explained by the changes in serum lipids and lipoproteins, and treatment with both estrogen and raloxifene independently predicted the response in aorta cholesterol. Because plasma levels of total raloxifene were low relative to clinical values in postmenopausal women, dose-response data for raloxifene are required. CONCLUSIONS: Our findings indicate that raloxifene hydrochloride has a potentially important antiatherogenic effect, analogous to that observed with estrogen in this model.

Hypocholesterolemic activity of raloxifene (LY139481): pharmacological characterization as a selective estrogen receptor modulator.

After once-daily oral dosing in ovariectomized rats, raloxifene (LY139481) hydrochloride produced dose- and time-dependent reductions in serum cholesterol and high-density lipoprotein-cholesterol. Paired-feeding studies demonstrated that effects of raloxifene on serum lipids were not secondary to effects on food consumption. Maximal reductions in serum cholesterol occurred within 4 days of raloxifene administration or sooner, depending on the administered dose. The ED50 for 50% reduction in serum cholesterol by raloxifene was 0.13 +/- 0.04 mg/kg/day (mean +/- S.E.M., n = 17); maximal cholesterol reduction by raloxifene (68%) was significantly less than that produced by estrogen (17 alpha-ethinylestradiol; 89%) after 4 to 7 days of daily dosing. Dose-response curves for cholesterol lowering by raloxifene were generated in the presence of varying doses of 17 alpha-ethinylestradiol; two-way analysis of variance revealed significant interactions between estrogen and raloxifene with respect to cholesterol lowering (P < .001). Furthermore, a high dose of raloxifene (10 mg/kg/day) prevented further reduction of serum cholesterol by estrogen (1-100 micrograms/kg/ day) beyond that produced by raloxifene alone. For a series of closely related structural analogs of raloxifene, log(ED50) values for cholesterol lowering were highly correlated with log(relative binding affinity) for the estrogen receptor (r = 0.93; P < .0001). Thus, cholesterol lowering by raloxifene in ovariectomized rats is mediated primarily via partial agonist effects at estrogen receptors. Taken together with previous observations in uterine tissue of estrogen antagonism by raloxifene in the absence of significant agonism, the present findings support the classification of raloxifene as a selective estrogen receptor modulator.

Inhibition of vascular smooth muscle cell proliferation and arterial intimal thickening by a novel antiproliferative naphthopyran.

Smooth muscle cell proliferation plays an important role in neointimal thickening after vascular injury and may contribute to restenosis after angioplasty. Development of suitable pharmacological agents modulating smooth muscle cell proliferation is critical for further investigation of vascular hyperplasia and its prevention. In the present study, we report a novel series of compounds that inhibit smooth muscle cell proliferation and arterial intimal thickening after balloon angioplasty. LY290181 (2-amino-4-[3-pyridyl]-4H-naphtho [1, 2-b]pyran-3-carbonitrile) and LY290293 (2-amino-4-[3-pyridyl]-4H-naphtho [1, 2-b]pyran-carbonitrile) produced a dose-dependent inhibition of DNA synthesis and proliferation of vascular smooth muscle cells in culture. Fifty percent inhibition (IC50) of cell proliferation was produced by 20 nM LY290181 or LY290293. Cell growth inhibition was not due to cell death, as demonstrated by the release of intracellular lactate dehydrogenase and by the reversibility of inhibition upon washing. Inhibition of smooth muscle cell proliferation was achieved in cells stimulated by either serum or individual growth factor such as platelet-derived growth factor, fibroblast growth factor or epidermal growth factor. In the rat model of balloon injury to carotid artery, LY290181 and LY290293 produced 61% (P < .005) and 48% (P < .005) inhibition of intimal thickening when administered p.o. at 100 and 120 mg/kg/day, respectively, over a 2-week period. Inhibition of intimal thickening (70%, P < .005) by LY290293 was also demonstrated when the compound was administered s.c. at 10 mg/kg/day. These studies demonstrate that naphthopyrans LY290181 and LY290293 are potent inhibitors of smooth muscle cell proliferation in vitro and that they produce substantial reduction in arterial intimal thickening in a balloon injury model when administered systemically.

Comparison of cocaine and opiate exposures between young urban and suburban children.

OBJECTIVE: To determine the prevalence of cocaine and opiate metabolites in the urine of young urban and suburban children. DESIGN: Survey. SETTING: Urban and suburban emergency departments and private pediatric practices. PATIENTS: A convenience sample of 1469 children between 1 and 60 months of age who required a urinalysis for investigation of the chief complaint. INTERVENTION: None. MAIN OUTCOME MEASURES: Urine was screened for benzoylecogonine and opiates using an enzyme-multiplied immunoassay technique and a fluorescence-polarization immunoassay, both with a sensitivity of 50 ng/mL. RESULTS: Benzoylecogonine was identified in the urine of 45 children (3.1%) (95% CI, 2.2% to 3.9%) and opiates in the urine of 38 children (2.6%) (95% CI, 1.8% to 3.4%). No difference was observed between urban and suburban health care facilities in the percentage of patients whose urine tested positive for benzoylecgonine (29 of 1011 vs 16 of 458, P = .6) or opiates (28 of 1011 vs 10 of 458, P = .6). CONCLUSION: Exposure to illicit drugs, as reflected by urinary metabolites, is similar for urban and suburban children.

Synthesis and biological evaluation of a new series of sterols as potential hypocholesterolemic agents.

A new series of sterols was synthesized and tested in a CHO cell-based LDL receptor/luciferase (LDLR/Luc) assay to investigate the capability of derepressing the transcription of LDL receptor promoter in the presence of 25-hydroxycholesterol. The effect of various substitutions on antagonizing the repressing effect mediated by 25-hydroxycholesterol was also studied in terms of regio- and stereochemistry, lipophilicity, steric bulk, and pi-electron density. Except 12, compounds active in the primary LDLR/Luc assay were not active in the secondary simian virus 40/luciferase (SV40/Luc) assay, demonstrating the specificity of their in vitro activity. Eight active compounds of various structural types were selected and screened in a [1-14C-acetate]cholesterol biosynthesis inhibition assay; none has shown any interference with the cholesterol biosynthesis in CHO cells. In hypercholesterolemic hamsters, generally, compounds that were active in vitro were active in vivo and vice versa, with the exception of three in vitro inactive compounds: 3 beta-ols 3a' and 3c' as well as 3-ketone 2a. Experimental results from the livers of hamsters revealed that the in vivo conversion of 3a' or 2a to 3a has in part contributed to the observed in vivo activity, and it is also anticipated that 3c' may similarly be converted to 3c in hamsters.

Inhibition of cholesteryl ester transfer protein in normocholesterolemic and hypercholesterolemic hamsters: effects on HDL subspecies, quantity, and apolipoprotein distribution.

The effects of cholesteryl ester transfer protein (CETP) inhibition on the serum lipoprotein profile in both normocholesterolemic and hypercholesterolemic hamsters has been determined following subcutaneous injection of 12.5 mg/kg of the CETP neutralizing monoclonal antibody, TP2. Inhibition of CETP activity was greater than 60% and resulted in a 30-40% increase in high density lipoprotein (HDL) in both normal and hypercholesterolemic animals. These HDL effects were observed 1 day post-injection, were maximal by 4 days, and returned to control values by 14 days. Inhibition of CETP activity resulted in a decrease in both low density lipoprotein (LDL) and very low density lipoprotein (VLDL) cholesterol concomitant with HDL increase, and in hypercholesterolemic animals resulted in increased total serum cholesterol. In addition to the quantitative differences in LDL and HDL, there were significant increases in the size of the HDL, a shift to smaller LDL particles, and changes in apolipoprotein (apo) composition as evaluated by FPLC and Western blot analysis. Large apoA-I-poor and apoE-containing HDL became prevalent in hypercholesterolemic hamsters after CETP inhibition. In addition, the size of the CETP-containing HDL particles increased with inhibition of transfer activity. While these effects were apparent in normocholesterolemic animals, the changes in apolipoprotein distribution and HDL subspecies as detected on native gels were more significant in the hypercholesterolemic animals. The changes in the HDL profile and apolipoprotein distribution after CETP inhibition in hamsters were similar to those reported in CETP-deficient Japanese subjects, suggesting the utility of the hypercholesterolemic hamster as an in vivo model for the understanding of the lipoprotein changes associated with CETP inhibition.

Raloxifene (LY139481 HCI) prevents bone loss and reduces serum cholesterol without causing uterine hypertrophy in ovariectomized rats.

There is a medical need for an agent with the positive effects of estrogen on bone and the cardiovascular system, but without the negative effects on reproductive tissue. Raloxifene (LY139481 HCI) is a benzothiophene derivative that binds to the estrogen receptor and inhibits the effects of estrogen on the uterus. In an ovariectomized (OVX) rat model we investigated the effects of raloxifene on bone loss (induced by estrogen deficiency), serum lipids, and uterine tissue. After oral administration of raloxifene for 5 wk (0.1-10 mg/kg per d) to OVX rats, bone mineral density in the distal femur and proximal tibia was significantly greater than that observed in OVX controls (ED50 of 0.03-0.3 mg/kg). Serum cholesterol was lower in the raloxifene-treated animals, which had a minimal effective dose of 0.1 mg/kg and an approximate oral ED50 of 0.2 mg/kg. The effects of raloxifene on bone and serum cholesterol were comparable to those of a 0.1-mg/kg per d oral dose of ethynyl estradiol. Raloxifene diverged dramatically from estrogen in its lack of significant estrogenic effects on uterine tissue. Ethynyl estradiol produced a marked elevation in a number of uterine histologic parameters (e.g., epithelial cell height, stromal eosinophilia). These data suggest that raloxifene has promise as an agent with beneficial bone and cardiovascular effects in the absence of significant uterine effects.

Vascular serotonin 5-hydroxytryptamine2 receptor blockade by LY215840: lack of effect upon neointima formation in balloon-injured rat carotid arteries.

5-Hydroxytryptamine (5-HT) is mitogenic for vascular smooth muscle cells, an effect that may result from 5-HT2 receptor activation. After arterial balloon injury, platelet-derived products including 5-HT are delivered to the denuded vessel wall via platelet adhesion/aggregation and degranulation. To assess the role of 5-HT2 receptors in neointima formation, the selective 5-HT2 receptor antagonist LY215840 was infused continuously in rats with osmotic minipumps from 2 days before until 2 weeks after balloon injury to the left common carotid artery, at which time effects upon intimal thickening were examined. Chronic i.v. infusion of LY215840 (25 micrograms/kg/hr) produced a significant, rightward shift (approximately 500-fold) in the 5-HT pressor-response curve in pithed rats determined at 1 hr, 4 days or 16 days after initiation of infusion. Under these conditions, LY215840 had no significant effect upon cross-sectional intimal area determined 2 weeks postballoon injury (0.118 +/- 0.010 vs. 0.114 +/- 0.017 mm2 for vehicle- and LY215840-treated groups, respectively). Furthermore, the cellular density of neointima from LY215840-treated rats was unchanged relative to that of the vehicle group. As a positive control, heparin infusion produced dose-dependent reductions in intimal thickening in this model. At the infusion rate of 0.22 mg/kg/hr, heparin reduced intimal area from a control value of 0.114 +/- 0.015 to 0.025 +/- 0.007 mm2 (P < .05). Thus, extensive blockade of vascular 5-HT2 receptors does not inhibit neointima formation in this model of vascular balloon injury.

Losartan, a nonpeptide angiotensin II (Ang II) receptor antagonist, inhibits neointima formation following balloon injury to rat carotid arteries.

Angiotensin-converting enzyme inhibitors have been shown to inhibit intimal thickening following balloon catheterization of rat carotid arteries. To assess the role of the renin-angiotensin pathway and the angiotensin type-I (AT1) receptor in this effect, the nonpeptide Ang II antagonist losartan (DuP 753) or vehicle was infused continuously i.v. in rats from two days before to two weeks after balloon injury to the left common carotid artery; drug effects upon intimal thickening were examined histologically. Losartan produced a dose-dependent reduction in cross-sectional area of intimal lesions determined two weeks post balloon injury. At 5 mg/kg/day a nonsignificant 23% reduction of intimal area was observed. At the higher dose of 15 mg/kg/day, losartan produced a 48% reduction in intimal area (P less than 0.05) compared to the vehicle-infused group. The cellular density of the neointima was not affected by losartan, indicating a probable effect of the drug upon migration and/or proliferation of smooth muscle cells. In separate groups of non-ballooned rats, losartan infusions of 5 and 15 mg/kg/day produced significant rightward shifts (averaging 6.4- and 55-fold, respectively) in curves relating increases in blood pressure to intravenous Ang II in pithed rats determined between 2 and 16 days following initiation of losartan infusion. Mean arterial blood pressure (determined under alpha-chloralose anesthesia) was reduced following continuous losartan infusion for 6 days from 128 +/- 8 mm Hg (vehicle) to 105 +/- 8 mm Hg at 5 mg/kg/day (P less than 0.05), and 106 +/- 4 mm Hg at 15 mg/kg/day (P less than 0.05). Thus, losartan attenuated the vascular response to balloon catheter injury, and this effect was associated with functional block of vascular AT1 receptors. The results support a role for Ang II, acting via AT1 receptors, in myointimal thickening subsequent to balloon injury of rat carotid arteries.

Cardiac inotropic responses to calcium and forskolin are not altered by prolonged isoproterenol infusion.

Effects of prolonged isoproterenol infusion upon the density of cardiac calcium channels, calcium-mediated contractile responses, and the ability of forskolin to enhance tension development and cyclic AMP accumulation were studied in ventricular muscle preparations from Sprague-Dawley rats. Isoproterenol infusion (400 micrograms/kg per h s.c., 4 days) significantly decreased calcium channel density (Bmax) in cardiac microsomal membranes as quantified by a 32% decrease in specific [3H]nitrendipine binding sites; binding affinity (KD) was unchanged. A 57% decrease of beta-adrenoceptors confirmed homologous down regulation. To examine functional effects of decreased [3H]nitrendipine binding sites, responses to calcium, BAY K8644 and nifedipine were determined in isolated right ventricular strips. Significant decreases in basal developed tension were observed in muscles from isoproterenol-infused rats. However, concentration-dependent increases in contractility in response to CaCl2 or BAY K8644 were comparable, and the negative inotropic effect of nifedipine was unchanged. Whereas isoproterenol infusion was associated with significantly decreased basal cardiac cyclic AMP concentrations, exposure of ventricular strips from either vehicle- or isoproterenol-infused rats to 10 microM forskolin resulted in comparable increases in cyclic AMP and in developed tension. Cumulative, submaximal concentrations of forskolin also produced similar increases in contractility with maximum responses in ventricular strips from vehicle-infused animals attained at 4.4 microM forskolin. Higher concentrations resulted in automaticity. By contrast, ventricle from isoproterenol-infused animals responded to 14.4 microM forskolin with maximal increases in force of contraction.

LY249933: a cardioselective 1,4-dihydropyridine with positive inotropic activity.

Compound LY249933 and its component diastereomers, (RR) and (SR), were studied for their vascular and cardiac effects in vitro and in vivo. In guinea pig cardiac ventricular membranes, LY249933, (RR), and (SR) potently displaced bound [3H]nitrendipine (Kd values = 2-6 nM). In isolated guinea pig right ventricular strips, LY249933 produced a small but significant increase in contraction, whereas (RR) substantially increased (-log EC50 (M) = 4.6 +/- 0.8) and (SR) decreased contraction (-log EC50 (M) = 4.1 +/- 0.8). In isolated canine cephalic vein, contracted with 80 mM KCl, an increase in contraction was produced by (RR), whereas relaxation was produced by LY249933 (-log EC50 (M) = 5.9 +/- 0.9) and (SR) (-log EC50 (M) = 6.0 +/- 0.7). At 20 mM KCl, (RR) increased, (SR) decreased, but LY249933 did not alter contraction. In anesthetized dogs, LY249933 (200 micrograms/kg/min, i.v.) increased dP/dt60, decreased heart rate, but did not change vascular resistance or rate pressure product. At the same dose, (RR) and (SR) both tended to increase dP/dt60 nonsignificantly, whereas (RR) increased and (SR) decreased vascular resistance. Both (RR) and (SR) tended to decrease heart rate nonsignificantly, whereas (RR) did not change and (SR) decreased rate pressure product. Thus, LY249933 produced potentially beneficial cardiovascular changes resulting from the combined actions of its (RR) and (SR) diastereomers that are postulated to be calcium agonist and antagonist, respectively.

Characterization and pharmacological relevance of high affinity binding sites for [3H]LY186126, a cardiotonic phosphodiesterase inhibitor, in canine cardiac membranes.

[3H]LY186126, an analogue of the cardiotonic agent indolidan, was shown to bind reversibly and with high affinity (Kd = 4 nM) to a single class of binding sites within canine myocardial vesicles. Binding site density measured in various cardiac membrane fractions correlated well with Ca2+-ATPase activity (r = 0.94; p less than 0.01), but not with Na+,K+-ATPase or azide sensitive ATPase, indicating a localization of these sites within sarcoplasmic reticulum membranes. Divalent cations were required for binding and displayed the following order of activation: Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+. Differential activation of [3H]LY186126 binding by various divalent cations was due to alterations in binding site density, rather than affinity. cGMP and selective inhibitors of type IV membrane-bound phosphodiesterase (SR-PDE), for example, indolidan, milrinone, imazodan, and enoximone, selectively displaced bound [3H]LY186126 caffeine, theophylline, and rolipram were relatively impotent as inhibitors of radiolabel binding. Kd values from displacement curves were highly correlated with IC50 values for inhibition of SR-PDE (r = 0.92; p less than 0.001). In addition, Kd values correlated well with published ED50 values for increases in cardiac contractility in pentobarbital-anesthetized dogs (r = 0.94; p less than 0.001). The results support the hypothesis that [3H]LY186126 labels the pharmacological receptor for the class of positive inotropic agents characterized as isozyme-selective phosphodiesterase inhibitors. Furthermore, the data suggest that the identity of the site labeled by [3H]LY186126 is SR-PDE, the type IV isozyme of cardiac phosphodiesterase located in the sarcoplasmic reticulum.

Synthesis of a tritium-labeled indolidan analogue and its use as a radioligand for phosphodiesterase-inhibitor cardiotonic binding sites.

We have radiolabeled a structural analogue of indolidan, a potent phosphodiesterase-inhibitor cardiotonic, to permit biochemical studies regarding the interaction of this class of drugs with their pharmacological receptor. [3H]-LY186126 (1,3-dihydro-3,3-dimethyl-1-[3H3]methyl-5-(1,4,5,6-tetrahydro-4-me thyl-6- oxo-3-pyridazinyl)-2H-indol-2-one; [3H]-3) was selected as a synthetic target because of its potency as a cardiotonic and the ability to readily incorporate three tritia via the indolone N-CH3 substituent. Alkylation of a desmethyl precursor with tritium-labeled iodomethane resulted in [3H]-3 with a radiochemical purity of 98% and a specific activity of 79.2 Ci/mmol. This radioligand binds with high affinity to myocardial membrane vesicles. The binding was saturable, and Kd and Bmax values of 4.1 nM and 383 fmol/mg protein were obtained. A series of indolidan congeners displaced [3H]-3 bound to myocardial vesicles, and Ki values for inhibition of binding were highly correlated with canine inotropic ED50 values, suggesting the specific binding of [3H]-3 to cardiac vesicles is pharmacologically relevant.