Matrix Metalloproteinases -14, -9 and -2 are Localized to the Podosome and Involved in Podosome Development in the A7r5 Smooth Muscle Cell.

AIM: The purpose of the study was to localize matrix metalloproteinase (MMP)-14, -9, and -2 in the A7r5 smooth muscle cell and to understand the interaction between these MMPs and the cytoskeleton. This interaction was observed under non-stimulating and phorbol 12, 13-dibutyrate (PDBu)-stimulating conditions. METHODS: Confocal microscopy was utilized to define the localizations of MMPs and tissue inhibitor of matrix metalloproteinases (TIMPs) in the A7r5 cell and to determine interaction between MMPs and the cytoskeleton. Under PDBu-stimulating conditions, the presence of MMP active forms and activity by gel zymography was evaluated in the A7r5 cell. Actin and microtubule-polymerization inhibitors were used to evaluate MMP interaction with the cytoskeleton and the cytoskeleton was observed on matrix and within a Type I collagen gel. RESULTS: MMP-14, -9, and -2 were localized to the podosome in the A7r5 smooth muscle cell and interactions were seen with these MMPs and the actin cytoskeleton. PDBu-stimulation induced increases in the protein abundance of the active forms of the MMPs and MMP-2 activity was increased. MMPs also interact with a-actin and not ß-tubulin in the A7r5 cell. Galardin, also known as GM-6001, was shown to inhibit podosome formation and prevented MMP localization to the podosome. This broad spectrum MMP inhibitor also prevented collagen gel contraction and prevented cell adhesion and spreading of A7r5 cells within this collagen matrix. CONCLUSION: MMPs are important in the formation and function of podosomes in the A7r5 smooth muscle cell. MMPs interact with a-actin and not ß-tubulin in the A7r5 cell. Podosomes play an important role in cell migration and understanding the function of podosomes can lead to insights into cancer metastasis and cardiovascular disease.

Diet-induced obesity: dopamine transporter function, impulsivity and motivation.

OBJECTIVE: A rat model of diet-induced obesity (DIO) was used to determine dopamine transporter (DAT) function, impulsivity and motivation as neurobehavioral outcomes and predictors of obesity. DESIGN: To evaluate neurobehavioral alterations following the development of DIO induced by an 8-week high-fat diet (HF) exposure, striatal D2-receptor density, DAT function and expression, extracellular dopamine concentrations, impulsivity, and motivation for high- and low-fat reinforcers were determined. To determine predictors of DIO, neurobehavioral antecedents including impulsivity, motivation for high-fat reinforcers, DAT function and extracellular dopamine were evaluated before the 8-week HF exposure. METHODS: Striatal D2-receptor density was determined by in vitro kinetic analysis of [(3)H]raclopride binding. DAT function was determined using in vitro kinetic analysis of [(3)H]dopamine uptake, methamphetamine-evoked [(3)H]dopamine overflow and no-net flux in vivo microdialysis. DAT cell-surface expression was determined using biotinylation and western blotting. Impulsivity and food-motivated behavior were determined using a delay discounting task and progressive ratio schedule, respectively. RESULTS: Relative to obesity-resistant (OR) rats, obesity-prone (OP) rats exhibited 18% greater body weight following an 8-week HF-diet exposure, 42% lower striatal D2-receptor density, 30% lower total DAT expression, 40% lower in vitro and in vivo DAT function, 45% greater extracellular dopamine and twofold greater methamphetamine-evoked [(3)H]dopamine overflow. OP rats exhibited higher motivation for food, and surprisingly, were less impulsive relative to OR rats. Impulsivity, in vivo DAT function and extracellular dopamine concentration did not predict DIO. Importantly, motivation for high-fat reinforcers predicted the development of DIO. CONCLUSION: Human studies are limited by their ability to determine if impulsivity, motivation and DAT function are causes or consequences of DIO. The current animal model shows that motivation for high-fat food, but not impulsive behavior, predicts the development of obesity, whereas decreases in striatal DAT function are exhibited only after the development of obesity.

Enhanced vascular contractility and diminished coronary artery flow in rats made hypertensive from diet-induced obesity.

OBJECTIVE: To determine whether obesity-induced hypertension was associated with alterations in vascular contractility and/or cardiac function. DESIGN: Male Sprague-Dawley rats were fed either a low fat (LF; 11% kcal as fat) or a moderately high fat (MHF; 32% kcal as fat) diet for 11 weeks. MEASUREMENTS: Body weight; mean arterial pressure; angiotensin peptides; mesenteric contractile response to phenylephrine (PE), potassium chloride (KCl), serotonin, angiotensin II (AngII), calcium chloride; baseline and isoproterenol-induced cardiac contractility; baseline and isoproterenol-induced coronary artery blood flow. RESULTS: Rats fed the MHF diet segregated into obesity-prone (OP) and obesity-resistant (OR) groups. OP rats exhibited elevations in mean arterial pressure (MAP) and elevations in systemic concentrations of angiotensin peptides. Mesenteric arteries from OP rats exhibited a greater contractile response to PE, KCl and serotonin (5-HT). Heightened responses to PE persisted in arteries from OP rats even after normalization of the response to KCl. In contrast, the response of permeabilized mesenteric arteries to a maximal concentration of calcium was similar in rats from each group. Isolated perfused hearts exhibited similar baseline and isoproterenol-induced contractility in rats from each group. However, isoproterenol was unable to increase coronary artery blood flow in hearts from OP rats. CONCLUSION: Enhanced vascular reactivity may contribute to obesity-induced hypertension, while reductions in coronary artery relaxation would impair the ability of the heart to respond to increased myocardial demand.

Effects of nornicotine enantiomers on intravenous S(-)-nicotine self-administration and cardiovascular function in rats.

RATIONALE: Previous neurochemical evidence indicates that R(+)-nornicotine is more potent than S(-)-nornicotine in evoking dopamine release in rat nucleus accumbens slices. OBJECTIVE: The current study tested the hypothesis that R(+)-nornicotine is also more potent than S(-)-nornicotine in selectively decreasing intravenous S(-)-nicotine self-administration in rats. RESULTS: After acute pretreatment (1-10 mg/kg for each enantiomer), R(+)-nornicotine was more potent than S(-)-nornicotine in decreasing S(-)-nicotine self-administration; in contrast, within the same dose range, the nornicotine enantiomers were equipotent in decreasing sucrose-maintained responding. This enantioselectivity does not likely reflect a difference in bioavailability, since similar levels of nornicotine were recovered from the brain 60 min after injection (5.6 mg/kg for each enantiomer). With repeated pretreatment, tolerance did not develop to the rate-decreasing effect of either nornicotine enantiomer (3 or 5.6 mg/kg) with respect to the decrease in S(-)-nicotine self-administration, although the enantioselectivity dissipated across repeated pretreatments. While both enantiomers acutely produced a similar increase in blood pressure and heart rate, tolerance developed to the blood pressure effects of R(+)-nornicotine, but not to the effects of S(-)-nornicotine, across repeated treatments. CONCLUSION: Both R(+)- and S(-)-nornicotine may have potential utility as a novel tobacco use cessation agent.

Antagonism of AT2 receptors augments angiotensin II-induced abdominal aortic aneurysms and atherosclerosis.

1. We have recently demonstrated that chronic infusion of Angiotensin II into apoE-/- mice promotes the development of abdominal aortic aneurysms. To determine the involvement of specific Angiotensin II receptors in this response, we co-infused Angiotensin II (1000 ng kg(-1) min(-1) for 28 days) with losartan (30 mg kg(-1) day(-1)) or PD123319 (3 mg kg(-1) day(-1)) to antagonize AT1 and AT2 receptors, respectively. 2. Infusion of Angiotensin II promoted the development of abdominal aortic aneurysms in 70% of mature female apoE-/- mice. The formation of aortic aneurysms was totally inhibited by co-infusion of Angiotensin II with losartan (30 mg kg(-1) day(-1); P=0.003). In contrast, the co-infusion of Angiotensin II with PD123319 resulted in a marked increase in the incidence and severity of aortic aneurysms. 3. To determine whether AT2 antagonism also promoted Angiotensin II-induced atherosclerosis, Angiotensin II was infused into young female apoE-/- mice that had little spontaneous atherosclerosis. In these mice, co-infusion of PD123319 led to a dramatic increase in the extent of atherosclerosis. This increase was associated with no change in plasma lipid concentrations and only transient and modest increases in blood pressure during co-infusion with PD123319. 4. While antagonism of AT1 receptors totally prevented the formation of aneurysms, antagonism of AT2 receptors promoted a large increase in the severity of Angiotensin II-induced vascular pathology.

Renin-angiotensin system and sympathetic nervous system in cardiac pressure-overload hypertrophy.

Angiotensin II and norepinephrine (NE) have been implicated in the neurohumoral response to pressure overload and the development of left ventricular hypertrophy. The purpose of this study was to determine the temporal sequence for activation of the renin-angiotensin and sympathetic nervous systems in the rat after 3-60 days of pressure overload induced by aortic constriction. Initially on pressure overload, there was transient activation of the systemic renin-angiotensin system coinciding with the appearance of left ventricular hypertrophy (day 3). At day 10, there was a marked increase in AT(1) receptor density in the left ventricle, increased plasma NE concentration, and elevated cardiac epinephrine content. Moreover, the inotropic response to isoproterenol was reduced in the isolated, perfused heart at 10 days of pressure overload. The affinity of the beta(2)-adrenergic receptor in the left ventricle was decreased at 60 days. Despite these alterations, there was no decline in resting left ventricular function, beta-adrenergic receptor density, or the relative distribution of beta(1)- and beta(2)-receptor sites in the left ventricle over 60 days of pressure overload. Thus activation of the renin-angiotensin system is an early response to pressure overload and may contribute to the initial development of cardiac hypertrophy and sympathetic activation in the compensated heart.

Presynaptic modulation of sympathetic neurotransmission in rat left ventricle slices: effect of pressure overload.

The purpose of this study was to establish the rat left ventricle (LV) tissue slice system for examination of norepinephrine (NE) release from sympathetic nerve terminals. Moreover, initial experiments were performed to use the LV tissue slice system to examine differences in NE uptake and release following cardiac pressure overload induced by abdominal aortic constriction (AC). Kinetic parameters (Vmax, Km) for the specific uptake of [3H]-NE demonstrated high affinity (Km, 1.94 +/- 0.83 microM) and moderate capacity uptake (Vmax, 182 +/- 6 fmol/mg/weight/min). Following 10 days of pressure overload, the Vmax for [3H]-NE uptake was significantly reduced (by 46%) in LV slices from AC rats compared to sham-operated (SO) controls. In control rat LV slices preloaded with [3H]-NE, electrically evoked [3H]-overflow was calcium- and stimulus pulse number-dependent. The neuronal uptake inhibitor, desipramine (DMI), increased (by 60%) evoked [3H]-overflow from LV slices. The alpha2-agonist, UK14304, decreased evoked [3H]-overflow from LV slices in a concentration-dependent manner (maximal reduction of 75%). The beta2-agonist, salbutamol, increased evoked [3H]-overflow from LV slices in a concentration-dependent manner (maximal increase of 200%). In separate experiments, the LV tissue slice system was used to examine the effect of pressure overload on evoked [3H]-overflow. Following 10 days of pressure overload, evoked [3H]-overflow from LV slices of AC rats was increased (by 50%) compared to SO control. Increases in evoked [3H]-overflow from LV slices of AC rats compared to SO controls remained evident in the presence of DMI. These results demonstrate the relative importance of NE release and uptake using an in vitro LV tissue slice system. Sympathetic nerve terminals innervating rat LV were demonstrated to possess functional presynaptic alpha2- and beta2-adrenergic receptors. Finally, using this LV tissue slice system, reductions in the uptake velocity and increases in evoked NE release were demonstrated in response to acute cardiac pressure overload.

Fat cell metabolism: insulin, fatty acids, and renin.

Risk factors contributing to the potential inter-relationship between obesity and hypertension include insulin, fatty acids, and angiotensin II. All of these mediators are either produced by or act on adipocytes, influence fat cell metabolism, and have effects on the cardiovascular system. Moreover, these three mediators have several potential sites for positive feedback interaction, thus exacerbating the influence of any single risk factor. The purpose of this review is to highlight recent advances in our understanding of the influence of insulin, fatty acids, and angiotensin II on fat cell metabolism. Special emphasis is placed on potential adipose-related mechanisms of these factors, which would predictably elevate blood pressure. Given the prevalence of obesity and hypertension in the American population, delineation of potential pharmacologic targets that would influence both of these disease states is of primary importance to the successful treatment of these diseases of the metabolic syndrome X.

Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice.

Increased plasma concentrations of angiotension II (Ang II) have been implicated in atherogenesis. To examine this relationship directly, we infused Ang II or vehicle for 1 month via osmotic minipumps into mature apoE(-/-) mice. These doses of Ang II did not alter arterial blood pressure, body weight, serum cholesterol concentrations, or distribution of lipoprotein cholesterol. However, Ang II infusions promoted an increased severity of aortic atherosclerotic lesions. These Ang II-induced lesions were predominantly lipid-laden macrophages and lymphocytes; moreover, Ang II promoted a marked increase in the number of macrophages present in the adventitial tissue underlying lesions. Unexpectedly, pronounced abdominal aortic aneurysms were present in apoE(-/-) mice infused with Ang II. Sequential sectioning of aneurysmal abdominal aorta revealed two major characteristics: an intact artery that is surrounded by a large remodeled adventitia, and a medial break with pronounced dilation and more modestly remodeled adventitial tissue. Although no atherosclerotic lesions were visible at the medial break point, the presence of hyperlipidemia was required because infusions of Ang II into apoE(+/+) mice failed to generate aneurysms. These results demonstrate that increased plasma concentrations of Ang II have profound and rapid effects on vascular pathology when combined with hyperlipidemia, in the absence of hemodynamic influences.

Cold exposure regulates the norepinephrine uptake transporter in rat brown adipose tissue.

The neuronal uptake of norepinephrine (NE) in sympathetically innervated tissues is mediated by a high-affinity NE uptake transporter (NET). Rat interscapular brown adipose tissue (ISBAT) is densely innervated by the sympathetic nervous system for the control of cold- and diet-induced thermogenesis. To determine if cold exposure regulates the NET, kinetic parameters for [3H]NE uptake and [3H]nisoxetine (Nis) binding were determined in ISBAT from 7-day cold-exposed (CE) and control rats. Uptake of [3H]NE in ISBAT slices was of high affinity (1.6 microM). After 7 days of cold exposure the affinity for [3H]NE uptake was not altered; however, the uptake capacity was decreased (38%) in ISBAT slices from CE rats. Kinetic parameters for [3H]Nis binding demonstrated a single high-affinity site in ISBAT from CE and control rats with similar affinity. The density of [3H]Nis sites in ISBAT was decreased (38%) following cold exposure. A time course (2 h-7 days) for cold exposure demonstrated downregulation of [3H]Nis binding density by day 3, which remained through day 7. The affinity for [3H]Nis binding was transiently decreased at 2 h of cold exposure. Similarly, ISBAT NE content was decreased at 2 h of cold exposure. Pair feeding CE rats to food intake of controls normalized plasma NE content; however, [3H]Nis binding density in ISBAT remained decreased in pair-fed rats. These results demonstrate that the ISBAT NET is downregulated following cold exposure. Reductions in ISBAT NE content precede alterations in NET density; however, plasma NE content is not related to regulation of the NET.

Mechanisms contributing to angiotensin II regulation of body weight.

Previous studies in our laboratory have implicated adipose tissue as a potential site for local angiotensin II (ANG II) synthesis. However, functions of ANG II in adipose tissue and the impact of ANG II on body weight regulation are not well defined. To study the effect of ANG II on body weight, a chronic ANG II infusion model was used. In study 1, a low dose of ANG II (175 ng.kg-1.min-1) was infused into rats for 14 days. Plasma ANG II levels were not elevated after 14 days of infusion. ANG II-infused rats did not gain weight over the 14-day protocol and exhibited a lower body weight than controls on day 8. Food intake was not altered, but water intake was increased in ANG II-infused rats. Blood pressure gradually increased to significantly elevated levels by day 14. Thermal infrared imaging demonstrated an increase in abdominal surface temperature. Measurement of organ mass demonstrated site-specific reductions in white adipose tissue mass after ANG II infusion. In study 2, the dose-response relationship for ANG II infusion (200, 350, and 500 ng.kg-1.min-1) was determined. Body weight (decrease), blood pressure (increase), white adipose mass (decrease), plasma ANG II levels (increase), and plasma leptin levels (decrease) were altered in a dose-related manner after ANG II infusion. In study 3, the effect of ANG II infusion (350 ng.kg-1.min-1) was examined in rats treated with the vasodilator hydralazine. Hydralazine treatment normalized blood pressure in ANG II-infused rats. The effect of ANG II to decrease body weight was augmented in hydralazine-treated rats. These results demonstrate that low levels of ANG II infusion regulate body weight through mechanisms related to increased peripheral metabolism and independent of elevations in blood pressure.

Near-IR and IR imaging in lipid metabolism and obesity.

Approximately one-third of Americans are classified as obese. There has long been an interest in drug therapies for obesity. Interest in obesity research and in drug interventions in obesity has greatly increased since the discovery of a protein named leptin, one of apparently many competing biological signals in energy metabolism. The complexity of the obesity problem demands new non-invasive and non-destructive methods for monitoring lipid metabolism and energy expenditure to study the competing biological signals and their effects. A new computer algorithm for spectrometric imaging of living subjects is used to remove artifacts arising from subject motion from spectra and images. The algorithm is sufficiently simple to be implemented easily in hardware for real-time video processing. Because the algorithm can be applied to images, thermogenesis and lipid metabolism in interscapular adipose tissue can be observed directly in unrestrained and unanesthetized subjects using an InSb focal plane array video camera. The accuracy and precision of temperature and spectral measurements are established using laboratory references and prototype drugs in test subjects.

Characterization and regulation of angiotensin II receptors in rat adipose tissue. Angiotensin receptors in adipose tissue.

Characterization and regulation of angiotensin II (AII) receptor binding sites was performed in rat membrane preparations from nonadipose (liver, lung) and adipose (interscapular (ISBAT) and periaortic (PA) brown adipose tissue; epididymal (EF) and retroperitoneal (RPF) white adipose tissue). In membrane preparations from brown and white adipose sources, [125I]AII saturation binding revealed a single, high affinity (Kd range of 0.3 -0.6 nM) binding site with a modest AII receptor density (Bmax range of 17-120 fmol/mg protein) comparable to rat lung (130 fmol/mg protein). White adipose tissue contained a greater number of AII receptor sites than brown adipose tissue. Competition displacement studies demonstrated the AT1 receptor is the only angiotensin receptor subtype localized in adipose tissue, with the rank order for competition of [125I]AII binding in all adipose tissues examined AIII > AII > losartan > angiotensin I (AI) > PD123319. The AT2 specific receptor antagonist, PD123319, was ineffective at displacing [125I]AII binding in all adipose tissues examined. Since components of the renin-angiotensin system are regulated in adipose tissue, we determined if the AII receptor is also regulated in the obese state. AII receptor binding characteristics were determined in liver, lung, ISBAT and EF membrane preparations from adult Zucker obese (fa/fa) and lean (Fa/?) rats. AII receptor density was decreased in liver from obese rats. In contrast, the affinity for [125I]AII binding was not altered in tissues from obese rats. In a separate group of obese and lean rats, regulation of the AII receptor by phenobarbital (PB) was examined. Administration of PB restored AII receptor density in liver from obese rats to levels obtained in lean rats. In summary, these results demonstrate the presence of AT1 receptor sites in brown and white adipose tissue. Moreover, AII receptor density is decreased in tissues from obese rats, with restoration of receptor density by administration of PB. Future studies will determine if PB regulates the AT1 receptor at the level of gene expression.

Angiotensin II in brown adipose tissue from young and adult Zucker obese and lean rats.

Previous studies demonstrated that interscapular brown adipose tissue (ISBAT) produces angiotensin II (ANG II), which facilitates sympathetic neurotransmission (SN). ANG II content and regulation of SN were examined in young (17 days) and adult (16 wk) Zucker obese and lean rats. ANG II content in ISBAT from preobese rats was decreased compared with lean littermates. Evoked 3H overflow in ISBAT slices preloaded with [3H]NE was greater in preobese rats compared with control. ANG II increased evoked 3H overflow in ISBAT slices to a greater extent in preobese rats compared with control. [3H]NE uptake in ISBAT slices from preobese rats was decreased compared with control. In adult obese rats, plasma renin activity was decreased compared with control. ISBAT ANG II content was increased in adult obese rats compared with control. Evoked 3H overflow in ISBAT slices preloaded with [3H]NE was not different between obese and control. ANG II did not increase evoked 3H overflow in obese rats; however, ANG II increased evoked 3H overflow in lean rats. [3H]NE uptake in ISBAT slices from obese rats was decreased compared with control. These results suggest that ANG II modulation of SN activity is decreased in ISBAT from adult obese rats. In contrast, in young obese rats, increased SN activity and ANG II regulation of SN were evident in brown adipose tissue.

Acute and chronic losartan administration: effect on angiotensin II content and modulation of [3H]norepinephrine release from rat interscapular brown adipose tissue.

To determine if acute or chronic (21 days) losartan (10 mg/kg, s.c.) regulates the renin-angiotensin system in interscapular brown adipose tissue, angiotensin II (AII) content and [3H]overflow from slices preloaded with [3H]norepinephrine were examined. Acute or chronic losartan administration had no effect on AII content. AII increased evoked [3H] overflow from slices from control rats. Losartan administration did not alter basal [3H]outflow or evoked [3H]overflow. Acute losartan administration inhibited AII-induced enhancement of evoked [3H]overflow. Tolerance developed to the inhibitory effect of losartan following chronic administration.

Role of angiotensin II in brown adipose thermogenesis during cold acclimation.

The role of angiotensin II (ANG II) in increased sympathetic neuroeffector mechanisms observed in cold-induced thermogenesis of brown adipose tissue (BAT) was examined. Cold exposure (4 degrees C) for 7 days resulted in an increase in interscapular fat (ISF) ANG II content expressed per gram wet weight or per lobe of ISF, without concomitant changes in plasma components of the renin-angiotensin system. Additionally, in ISF slices preloaded with [3H]norepinephrine (NE), ANG II (10 nM) resulted in an increase (3-fold) in evoked 3H overflow from ISF slices from cold-exposed rats compared with ambient temperature controls. However, although basal 3H outflow was increased (2-fold) in ISF slices from cold-exposed rats, evoked 3H overflow was not different between ISF slices from cold-exposed and control rats. Specific neuronal uptake of [3H]NE in ISF slices from cold-exposed rats was decreased by 64%. Administration of the non-peptide AT1-receptor antagonist losartan to cold-exposed rats resulted in complete inhibition of ANG II-mediated presynaptic facilitation of evoked 3H overflow from ISF slices. However, losartan administration had no effect on cold-induced increases in ANG II content, protein content, and decreases in neuronal [3H]NE uptake in ISF. Results from these studies suggest that cold-induced thermogenesis of BAT results in alterations in presynaptic ANG II facilitation of NE release and defects in removal of NE from the synaptic cleft (neuronal uptake), both of which would enhance sympathetic nervous system-mediated thermogenesis. Furthermore, these results demonstrate a role for ANG II in enhanced sympathetic activity of cold-induced thermogenesis in BAT.

Near-IR imaging of atheromas in living arterial tissue.

A near-IR imaging system and parallel vector supercomputer are used with a fiber-optic probe to produce chemical maps of the intimal surface of living arteries. Spectrometric information collected at hundreds of near-IR wavelengths is assembled into color pictures of the lipoprotein and apolipoprotein composition of atheromas using a vectorized 3-D cellular automaton-based algorithm that operates in parallel. The nonparametric mathematics developed to identify and quantify the constituents of each voxel in the artery wall avoid the matrix factorizations that generate excess error in other pattern recognition methods and permit analysis in a wavelength space of over 1000 dimensions using fewer than 100 calibration samples. A surface feature resolution of 5.5 microns and depth resolution of 6.5 microns are achieved with the system. Data from the fiber optics confirm the injury hypothesis of lesion formation and the differing roles of HDL and LDL in cholesterol transport. In clinical studies, approximately 1/2 of human arterial lesions appear fibrous and contain little or no lipid. As such, these lesions would not be expected to regress in response to cholesterol-lowering agents such as lovastatin. Identification of lesion types in vivo will enhance the efficacy of treatment programs.

Acute and chronic effects of losartan (DuP 753) on blood pressure and vascular reactivity in normotensive rats.

The effects of in vivo treatment with the nonpeptide subtype 1 angiotensin II receptor antagonist, losartan, on blood pressure and vascular reactivity in normotensive male Sprague-Dawley rats were studied. Initial acute experiments demonstrated that blood pressure was significantly decreased six hours following a single injection of losartan (10 mg/kg, sc), but returned to control levels by 24 hours post-injection. Pressor responses to angiotensin II (0.1 ug/kg, iv) in these rats were significantly attenuated 2 and 24 hours following losartan injection. For chronic studies, rats were injected once daily for 21 days with either the same dose of losartan or saline vehicle. Blood pressure and pressor responses to angiotensin II were assessed at the end of the 21 day treatment period. A significant decrease in blood pressure was observed in chronic losartan treated rats 6 hours after the last injection on day 21; however, as in the acute studies, blood pressure had returned to control values by 24 hours post-injection. Although blood pressure had returned to normal, pressor responses to angiotensin II were significantly attenuated in chronic losartan treated rats 24 hours after the last injection. Following the in vivo studies, aortae and tail arteries were removed for experiments on vascular reactivity. Acute and chronic losartan treatment had no effect on KCl and norepinephrine reactivity. Endothelial-dependent and independent relaxation responses were also unaltered. A significant decrease in the maximal contractile response to angiotensin II was observed in aorta from acute and chronic losartan treated rats. Electrical stimulation-induced responses were unaltered in tail arteries from rats acutely treated with losartan but were potentiated in rats chronically treated with losartan. Exogenously applied angiotensin II, in concentrations which did not elicit contractile responses, potentiated electrical stimulation-induced responses of tail arteries from control rats but did not influence responses in arteries from acute and chronic losartan treated rats. These results demonstrate that losartan has significant blood pressure lowering effects in normotensive rats. Interestingly, although blood pressure returns to normal by 24 hours post-injection, pressor responses to angiotensin II remain attenuated in acute and chronic losartan treated rats. Finally, losartan treatment results in specific alterations in vascular reactivity associated with the actions of angiotensin II.

Angiotensin II and monocrotaline-induced pulmonary hypertension: effect of losartan (DuP 753), a nonpeptide angiotensin type 1 receptor antagonist.

Administration of the pyrrolizidine alkaloid monocrotaline (MCT) to rats results in hypertensive pulmonary vascular disease characterized by a structurally based increase in pulmonary vascular resistance and right ventricular hypertrophy. Alterations in lung angiotensin converting enzyme activity in MCT-treated rats have suggested a role for angiotensin II (AII) in the pathogenesis of this model of hypertensive pulmonary vascular disease. To determine if increases in AII contribute to the development of pulmonary hypertension in MCT-treated rats, we examined the effect of chronic administration of the nonpeptide AII receptor antagonist Losartan on indices of pulmonary hypertension, Losartan (DuP 753; 10 mg/kg s.c.) administration for 21 days did not prevent the development of hypertensive pulmonary vascular disease in MCT-treated rats. However, 18 hr after the last dose of Losartan, AII (0.1 micrograms/kg i.v.)-induced pressor responses were inhibited by 63% in Losartan-treated rats. Losartan administration in MCT-treated rats did not prevent increases in pulmonary artery pressure or development of right ventricular hypertrophy. Additionally, increases in medial arterial thickness in pulmonary artery vessels (less than 50 microns and 50-100 microns external diameter) from MCT-treated rats were still evident in Losartan-treated rats. However, Losartan administration decreased medial pulmonary artery thickness of 50 to 100 microns external diameter vessels in control rats. These results demonstrate that AII. acting at the AT1 receptor subtype, does not contribute to pulmonary hypertension in this animal model.