Dose-dependent therapeutic effects of 2-Methoxyestradiol on Monocrotaline-Induced pulmonary hypertension and vascular remodelling.

2-Methoxyestradiol (2ME) is a major non-oestrogenic metabolite of oestradiol. Our previous studies, performed in several models of cardiac and/or vascular injury, suggest that 2ME strongly inhibits both pressure-dependent and pressure-independent cardiac and vascular remodelling. Furthermore, recently we have shown that in male rats 2ME attenuates the development and retards the progression of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH); and in female rats 2ME eliminates the exacerbation of PAH and increased mortality due to ovariectomy. In the present study we compared the therapeutic effects of three different doses of 2ME (3, 10 and 30 microg/kg/hour; 2ME-3, 2ME-10 and 2ME-30, respectively) in male rats with MCT-induced PAH. The animals were also monitored for plasma 2ME levels and potential oestrogenic effects. Treatments were initiated 12 days after administration of MCT (60 mg/kg, i.p.). Twenty-eight days post MCT, right ventricular peak systolic pressure (RVPSP) was measured and morphometric analysis was conducted. All three doses of 2ME produced beneficial therapeutic effects in pulmonary hypertensive animals, i.e. reduced pulmonary artery pressure and right ventricular hypertrophy, attenuated pulmonary vascular remodelling and inflammatory response, and had favourable effects on survival. Notably, none of the three doses had any effect on plasma testosterone levels or on seminal vesicle or testicle weight. Dose-dependent increases in 2ME plasma levels were observed only with 2ME-3 and 2ME-10; 2ME-30 produced 2ME plasma levels similar to those seen with 2ME10. Nonetheless, 2ME-30 was significantly more efficacious than 2ME-3 or 2ME-10 and eliminated the high mortality (34%) induced by MCT. In summary, the present study indicates that 2ME, used in doses that produce plasma levels similar to those seen in the last trimester of pregnancy (1000-3000 pg/ml), is effective and safe (i.e. has no oestrogenic effects) in experimental PAH. These data also suggest that 2ME disposition, rather than plasma concentration, determines the therapeutic effects of 2ME in PAH.

2-Hydroxyestradiol attenuates the development of obesity, the metabolic syndrome, and vascular and renal dysfunction in obese ZSF1 rats.

A pandemic of obesity is contributing importantly to the prevalence of the metabolic syndrome characterized by hypertension, insulin resistance, and hyperlipidemia. In turn, the metabolic syndrome is contributing to vascular disease and the accelerating epidemic of chronic renal failure. Currently, pharmacological approaches to attenuate obesity and its cardiovascular/renal sequelae are limited. The purpose of this study was to determine the effects of 2-hydroxyestradiol, a metabolite of 17beta-estradiol with minimal estrogenic activity, on the development of obesity, the metabolic syndrome, and heart, vascular, and renal dysfunction in obese ZSF1 rats, a well-characterized genetic model of obesity and the metabolic syndrome with concomitant heart, vascular, and kidney disease. ZSF1 rats were treated, beginning at 12 weeks of age, for 26 weeks with vehicle or 2-hydroxyestradiol (10 microg/kg/h). At baseline and after 24 weeks of treatment, animals were placed in metabolic cages, and food intake, water intake, urine output, and urinary excretion of proteins and glucose were determined. Next, in fasting animals, plasma cholesterol was measured, an oral glucose tolerance test was conducted, and total glycated hemoglobin levels were determined. At the end of the study, animals were anesthetized and instrumented for assessment of heart performance, renal hemodynamics, and mesenteric vascular reactivity. 2-Hydroxyestradiol attenuated the development of obesity and improved endothelial function, decreased nephropathy, decreased the severity of diabetes, lowered arterial blood pressure, and reduced plasma cholesterol. 2-Hydroxyestradiol may be an important lead for the development of safe and effect drugs to attenuate obesity and its metabolic, vascular, and renal sequelae.

A(1) receptor blockade induces natriuresis with a favorable renal hemodynamic profile in SHHF/Mcc-fa(cp) rats chronically treated with salt and furosemide.

Our goal was to test the hypothesis that A(1) receptor blockade induces diuresis/natriuresis with a favorable renal hemodynamic/cardiac profile in aged, lean SHHF/Mcc-fa(cp) rats, a rodent model of hypertensive dilated cardiomyopathy. Thirteen-month-old SHHF/Mcc-fa(cp) rats were pretreated for 72 h before experiments with furosemide (100 mg/kg by gavage 72, 48, and 24 h before experiments) to mimic the clinical setting of chronic diuretic therapy and were given 1% NaCl as drinking water to reduce dehydration/sodium depletion. Animals were instrumented for measurement of systemic and renal hemodynamics, renal excretory function, and cardiac performance, and baseline values were obtained during a 30-min clearance period. Animals then received either vehicle (n = 9), BG9719 [the S-enantiomer of 1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)] xanthine (also called CVT-124)] (highly selective A(1) receptor antagonist; 0.1 mg/kg bolus + 10 microg/kg/min; n = 9) or furosemide (loop diuretic; 30 mg/kg; n = 8) and measurements were repeated during four subsequent clearance periods. Both BG9719 and furosemide increased urine volume and absolute and fractional sodium excretion. BG9719 increased renal blood flow and glomerular filtration rate, but did not affect fractional potassium excretion. Furosemide decreased renal blood flow and glomerular filtration rate and increased fractional potassium excretion. Neither drug altered afterload; however, furosemide, but not BG9719, decreased preload (central venous pressure and ventricular end diastolic pressure). Neither drug altered systolic function (+dP/dt(max)); however, furosemide, but not BG9719, attenuated diastolic function (decreased -dP/dt(max), increased tau). In the setting of left ventricular dysfunction, chronic salt loading and prior loop diuretic treatment, selective A(1) receptor antagonists are effective diuretic/natriuretic agents with a favorable renal hemodynamic/cardiac performance profile.

Inhibition of adenosine deaminase attenuates endotoxin-induced release of cytokines in vivo in rats.

The purpose of this study was to investigate in vivo the effects of modulating the adenosine system on endotoxin-induced release of cytokines and changes in heart performance and neurohumoral status in early, profound endotoxemia in rats. Time/pressure variables of heart performance and blood pressure were recorded continuously, and plasma levels of tumor necrosis factor alpha (TNFalpha), interleukin 1-beta (IL-1beta), plasma renin activity (PRA), and catecholamines were determined before and 90 min after administration of endotoxin (30 mg/kg of lipopolysaccharide, i.v.). Erythro-9[2-hydroxyl-3-nonyl] adenine (EHNA; an adenosine deaminase inhibitor) had no effects on measured time-pressure variables of heart performance under baseline conditions and during endotoxemia, yet significantly attenuated endotoxin-induced release of cytokines and PRA. Pretreatment with the non-selective adenosine receptor antagonist DPSPX not only prevented the effects of EHNA but also increased the basal release of cytokines and augmented PRA. At baseline, caffeine (a non-selective adenosine receptor antagonist) increased HR, +dP/dtmax, heart rate x ventricular pressure product (HR x VPSP) and +dP/dtmax normalized by pressure (+dP/dtmax/VPSP), and these changes persisted during endotoxemia. Caffeine attenuated endotoxin-induced release of cytokines and augmented endotoxin-induced increases in plasma catecholamines and PRA. Pretreatment with propranolol abolished the effects of caffeine on heart performance and neurohumoral activation during the early phase of endotoxemia. 6N-cyclopentyladenosine (CPA; selective A1 adenosine receptor agonist) induced bradicardia and negative inotropic effects, reduced work load (i.e., decreased HR, VPSP, +dP/dtmax, +dP/dtmax/VPSP and HR x VPSP) and inhibited endotoxin-induced tachycardia and renin release. CGS 21680 (selective A2A adenosine receptor agonist) decreased blood pressure under basal condition but did not potentiate decreases in blood pressure during endotoxemia. CGS 21680 completely inhibited endotoxin-induced release of TNFalpha, augmented sympathetic activity and PRA, and increased +dP/dtmax and +dP/dtmax/VPSP in the absence and presence of endotoxin. The present study provides strong evidence that inhibition of adenosine deaminase reduces cytokine release in vivo without producing significant hemodynamic and cardiac effects during the early phase of profound endotoxemia in rats. The augmented neurohumoral activation induced by caffeine is associated with decreased cytokine release induced by endotoxin. Further studies are warranted to determine the impact of these effects on cardiac function and hemodynamics in the late phase of endotoxemia.

Renal and metabolic effects of caffeine in obese (fa/fa(cp)), diabetic, hypertensive ZSF1 rats.

In Western society, the triad of hypertension, metabolic syndrome and obesity (which caries a high risk for renal disease) is increasing, as is the intake of caffeine. However, no information is available regarding the metabolic or renal consequences of caffeine consumption in this complex disease entity. The purpose of this study was to investigate the effects of chronic caffeine consumption on renal function and metabolic status in obese ZSF1 rats, an animal model of obesity, hypertension and the metabolic syndrome. Fifteen, 18-week-old male, obese ZSF1 rats were randomized to drink tap water (Cont, n = 8) or 0.1% solution of caffeine (Caff, n = 7) for 8 weeks. Metabolic and renal function measurements were performed at baseline and after 4 and 8 weeks of treatment. Caffeine treatment significantly (p < 0.05) reduced body weight, food, and fluid consumption and improved insulin sensitivity (fasting insulin 129.6+/-8.1 vs 97.5+/-3.6 microIU/mL; fed insulin 146.3+/-8.5 vs 110.6+/-3.4 microIU/mL; fasting glucose 138.7+/-13.4 vs 145+/-8.0 mg/dL; fed glucose 373+/-19.4 vs 283.3+/-19.6 mg/dL, Cont vs Caff, respectively). After 8 weeks of caffeine treatment, animals were less glycosuric as compared with control group. Area under glucose curves (AUC-glucose) in oral glucose tolerance test did not differ between the two groups (AUC- glucose: 592.5+/-42.7 vs 589.5+/-20.5 mg/dL x h, Cont vs Caff), whereas caffeine treatment significantly decreased AUC of insulin (AUC-insulin: 257.77+/-12.9 vs 198.0+/-5.9 microIU/mL x h, Cont vs. Caff, p<0.05). No differences were found with regard to plasma triglycerides and glycerol levels; however, caffeine significantly increased cholesterol levels after 4 and 8 weeks (2F-Anova, p<0.001). Moreover, caffeine significantly decreased creatinine clearance after 4 and 8 weeks (CrCl, Cont: 3.5+/-0.4, Caff: 2.2+/-0.2 L/kg/day, p<0.05) and increased protein/CrCl ratio (Cont: 323+/-30, Caff: 527+/-33 mg/L/day). Caffeine treatment for 8 weeks tended to increase plasma norepinephrine levels (p<0.06), but the two groups did not differ with regard to plasma renin activity, blood pressure, renal blood flow or and renal vascular resistance. The study indicates that caffeine improves insulin sensitivity but increases plasma cholesterol levels and impairs renal function in obesity with the metabolic syndrome and hypertension. Our results imply that the health consequences of chronic caffeine consumption may depend heavily on underlying pathophysiology process.

Inhibition of cytokine release by and cardiac effects of type IV phosphodiesterase inhibition in early, profound endotoxaemia in vivo.

1. In rats, inhibition of type IV phosphodiesterase (PDE4) attenuates acute renal failure and early (hours) mortality induced by high-dose endotoxin. Because it is unlikely that protection of renal function accounts for improved early survivability, most likely PDE4 inhibition exerts multiple beneficial effects in endotoxaemia and the purpose of the present study was to test this hypothesis. 2. In study 1, we determined, in anaesthetized rats, the effects of endotoxin (30 mg/kg, i.v.) on cardiac performance parameters (heart rate (HR), ventricular peak systolic pressure (VPSP), maximum positive change in left ventricular pressure with respect to time (+dP/dt), maximum negative change in left ventricular pressure with respect to time (-dP/dtmax), ventricular end-diastolic pressure (VEDP), ventricular minimum diastolic pressure (VMDP) and HR-pressure product), plasma catecholamine levels, plasma renin activity (PRA) and plasma levels of inflammatory cytokines (tumour necrosis factor (TNF)-alpha and interleukin (IL)-lbeta). 3. In study 2, we determined, in anaesthetized rats, whether inhibition of PDE4 attenuates lipopolysaccharide (LPS)-induced changes in the aforementioned parameters of heart performance and neurohumoral status. We compared the changes in these parameters induced by endotoxaemia in animals treated with either RO 20-1724 (10 microg/kg per min; a selective PDE4 inhibitor) or its vehicle (DMSO; 1.35 microL/min). 4. At 90 min postadministration, endotoxin significantly increased HR and reduced -dP/dtmax and VEDP and caused a several-fold increase in plasma levels of TNF-alpha, IL-1beta, noradrenaline, adrenaline and PRA. RO20-1724 significantly blunted the endotoxin-induced reduction in -dP/dtmax and decreased endotoxin-induced increases in TNF-alpha and IL-1beta without significantly altering endotoxin-induced changes in HR, VEDP, catecholamine levels and PRA. 5. Results from these studies indicate that, in addition to preserving renal function, PDE4 inhibition attenuates inflammatory cytokine release caused by high-dose endotoxin and may have protective effects on diastolic function in early profound endotoxaemia.

Renal function and structure in diabetic, hypertensive, obese ZDFxSHHF-hybrid rats.

The obese ZDFxSHHF-fa/fa(cp) model was developed by crossing lean female Zucker Diabetic Fatty (ZDF +/fa) and lean male Spontaneously Hypertensive Heart Failure (SHHF/Mcc-fa(cp), +/fa) rats. The purpose of the present study was to determine renal function and morphology, hemodynamics, and metabolic status in ZDFxSHHF rats. Two sets of experiments were conducted. First, we evaluated heart and kidney function and metabolic status in aged (46 weeks old) male obese ZDFxSHHF and age matched obese SHHF rats, lean Spontaneously Hypertensive (SHR) and lean normotensive Wistar Kyoto (WKY) rats. In the second set of experiments, renal function and structure as well as metabolic and lipid status were determined in lean (LN) and obese (OB) adult (29-weeks of age) ZDFxSHHF rats. At 46 weeks of age ZDFxSHHF rats are hypertensive expressing marked cardiac hypertrophy associated with diastolic dysfunction and preserved contractile function. Fasted hyperglycemia and hyperinsulinemia are accompanied by moderate hypercholesterolemia and hypertriglyceridemia. Obese aged ZDFxSHHF have marked renal hypertrophy, a 3-8 fold decrease in creatinine clearance (compared with SHHF, SHR and WKY), a high percent of segmental + global glomerulosclerosis (59.8%+/-10.8), and severe tubulointerstitial and vascular changes. Obese ZDFxSHHF rats die at an early age (approximately 12 months) from end-stage renal failure. Studies conducted in 29-week animals showed that, although both LN and OB 29-week old animals are hypertensive, OB animals have more severely compromised renal function and structure as compared with lean litter-mates (kidney weight: 2.56+/-0.16 vs. 1.61+/-0.12 g; creatinine clearance: 0.42+/-0.04 vs. 1.24+/-0.13 L/g kid/day; renal vascular resistance 12.39+/-1.4 vs. 6.14+/-0.42 mmHg/mL/min/g kid; protein excretion: 556+/-16 vs. 159+/-9mg/day/g kid, p < 0.05, OB vs. LN, respectively). Obesity is also associated with hyperglycemia (424+/-37 vs. 115+/-11 mg/dL), hyperinsulinemia (117.2+/-8.8 vs. 42.3+/-3.5 microU/mL), hypertriglyceridemia (5200+/-702 vs. 194+/-23 mg/dL), hypercholesterolemia (632+/-39 vs. 109+/-4mg/dL), and presence of segmental + global glomerulosclerosis (20.1+/-3.2% vs. 0.1+/-0.1%) with prominent tubular and interstitial changes (p < 0.05, OB vs. LN, respectively). In summary, the present study indicates that the crossing of rat strains of nephropathy produces hybrids that carry a high risk for severe renal dysfunction. The ZDFxSHHF rats express insulin resistance, hypertension, dislipidemia and obesity and develop severe renal dysfunction. In addition, the hybrids do not develop some of the complications (hydronephrosis or congestive heart failure) common for the parental strains that may compromise studies of renal function and structure. Therefore, the ZDFxSHHF rat may be a useful model fore valuating risk factors and pharmacological interventions in chronic renal failure.

Caffeine augments proteinuria in puromycin-aminonucleoside nephrotic rats.

Several studies indicate that increased intrarenal adenosine concentrations may attenuate puromycin-aminonucleoside (PAN)-induced nephropathy in rats. The purpose of this study was to investigate the chronic effects of caffeine, a nonselective adenosine receptor antagonist, on renal function and structure in PAN-induced nephropathy. Animals were randomized to receive drinking water or 0.1% caffeine solution. PAN was administered in two doses to a subset from each group at 1 week (100 mg/kg, s.c.; Purom-1) and 15 wks (80 mg/kg, s.c.; Purom-2) after initiating caffeine treatment (PAN and CAFF-PAN groups). The remaining animals served as time controls (CON and CAFF groups). Renal excretory function was followed for 23 wks. Caffeine consumption significantly augmented PAN-induced proteinuria after both PAN injections (Purom-1 and Purom-2, p<0.05 and p<0.001 respectively; CAFF-PAN vs. PAN). In addition, caffeine potentiated the transient reduction in creatinine clearance (CrCl) induced by PAN. Caffeine consumption for 23 wks significantly reduced CrCl in conscious nephrotic animals (4.76 +/- 0.98 vs. 8.51 +/- 1.55 L/kg/day, CAFF-PAN vs. PAN). Seven days after both PAN injections, increased plasma renin activity was detected in animals that were consuming caffeine as compared with corresponding control groups (CAFF and CAFF + PAN vs CON and PAN, respectively). Eight weeks after the second injection of PAN, acute measures of renal hemodynamic and excretory function were compared in anesthetized animals and renal samples were analyzed for histological changes. In PAN-rats, caffeine treatment for 23 weeks significantly reduced inulin clearance (0.28 +/- 0.09 vs. 0.57 +/- 0.12 mL/min/gr kidney. CAFF-PAN vs PAN, p<0.05), tended to increase renal vascular resistance (59.0 +/- 9.5 vs. 42.9 +/- 5.5 mmHg/mL/min/gr kidney, CAFF-PAN vs. PAN, p < 0.06), potentiated the development of more severe tubulointerstitial damage (tubular atrophy, presence of proteinaceous material, tubular dilatation, interstitial inflammation, interstitial fibrosis), and tended to increase glomerulosclerosis. In conclusion, this study indicates that caffeine adversely affects renal function in PAN-nephrotic rats, and that this effect may be due, in part, to increased activity of the renin angiotensin system.

Persistent improvement of cardiovascular risk factors in spontaneously hypertensive rats following early short-term captopril treatment.

This study was designed to determine whether an improvement in cardiovascular risk factors persists in spontaneously hypertensive rats (SHR) following withdrawal of angiotensin converting enzyme inhibitor (ACE-I) treatment. SHR were given deionized drinking water or captopril solution from four to sixteen weeks of age. At twelve weeks of age, rats from each group were instrumented with radiotelemetry devices for continuous monitoring of blood pressure. Mean arterial blood pressure was significantly lower in captopril-treated SHR during treatment (92+/-2 vs 147+/-1 mm Hg), and at twelve weeks after treatment withdrawal (131+/-2 vs 158+/-2 mm Hg). In addition, proteinuria, renal vascular resistance, plasma triglyceride levels, fasting glucose levels, post-prandial insulin levels, and heart weights were significantly reduced in the treated SHR compared to control SHR, at time-points between three to seven months after captopril withdrawal. Our findings indicate that short-term administration of an ACE-I during the developmental phase of hypertension in the SHR results in a long-term overall improvement of cardiovascular risk factors.

Effects of long-term caffeine consumption on renal function in spontaneously hypertensive heart failure prone rats.

Our previous studies supported the hypothesis that prolonged administration of caffeine to animals with high-renin hypertension causes progressive deterioration of renal function. However, thus far this hypothesis has been tested with only a few animal models of hypertension. The aim of this study was to test this hypothesis further by investigating the effects of long-term caffeine consumption on renal function in adult spontaneously hypertensive heart failure (SHHF/Mcc-fa(cp)) rats, another model of high-renin hypertension. Lean, male, 9-month-old SHHF/Mcc-fa(cp) rats were randomized to receive either normal drinking water (control group) or drinking water containing 0.1% caffeine (caffeine group) for 20 weeks. No changes in body weight, food and fluid intake, urine volume, and sodium and potassium excretion were found in conscious SHHF/Mcc-fa(cp) rats after 10 or 20 weeks of caffeine treatment. However, caffeine treatment accelerated the time-related decline in renal function and augmented urinary protein excretion. Ten weeks into the protocol, creatinine clearance was 3.6+/-0.4 and 5.7+/-0.9 L/kg/day in the caffeine group and control group, respectively (p<0.02), whereas 20 weeks into the study, creatinine clearance was similarly diminished in both groups. Proteinuria was greater in the caffeine group compared with the control group at both 10 (928+/-131 vs. 439+/-21 mg/kg/day, respectively; p<0.02) and 20 weeks (1,202+/-196 vs. 603+/-30 mg/kg/day, respectively; p<0.01) into the protocol. After 20 weeks, all animals were anesthetized and instrumented. Caffeine treatment for 20 weeks had no effects on blood pressure, heart rate, or vascular resistance in four examined vascular beds (abdominal aorta and renal, carotid, and mesenteric arteries). No changes in renal hemodynamics and electrolyte excretion were found, whereas significantly lower glomerular filtration rate (GFR; inulin clearance) and creatinine clearance (p<0.05) were observed in caffeine-treated animals. These data support our hypothesis that prolonged consumption of caffeine has adverse effects on renal function, in high-renin hypertension.

Caffeine increases renal renin secretion in a rat model of genetic heart failure.

In a previous study, we showed that caffeine (CAFF) increases basal renin secretion by blocking intrarenal adenosine receptors and, when sympathetic activity is increased, augments renin release in part by blockade of brain adenosine receptors, leading to increased central sympathetic tone. The purpose of this study was to investigate the effects of CAFF treatment on neurohumoral status and heart performance in experimental heart failure. Two series of experiments were performed. First, the effects of CAFF (10 mg/kg +150 microg/min over 40 min) on heart performance (time-pressure variables) and neurohumoral status were studied in conscious, 9-month-old Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHRs), and spontaneously hypertensive heart failure (SHHF/Mcc-fa(cp) rats. Second, caffeine (0.1% in drinking water) was given for 10 days to 14-month-old SHHF/Mcc-fa(cp) rats, and cardiac performance, renal function, and neurohumoral status determined in vivo. CAFF infusion increased heart rate, left ventricular peak systolic pressure, and workload in hypertensive (SHRs and SHHF), but not in normotensive (WKY) animals and had no effects on cardiac contractility in all three strains. CAFF increased plasma renin activity (PRA), norepinephrine (NE), and epinephrine (E) levels in all three strains [treatment effect, p<0.001, 2F analysis of variance (ANOVA)], and these effects were greater in hypertensive (SHRs and SHHF) animals as compared with normotensive WKY rats (p<0.015). Ten-day CAFF treatment in 14-month-old SHHF did not change measured cardiac time-pressure variables, or hemodynamic or renal excretory function parameters that can affect renin secretion. However, CAFF treatment significantly increased renal renin secretion (71.1+/-19.2 vs. 9.5+/-5.8 ng Ang I/h/min/kg for caffeine and control group, respectively; p<0.01). In summary, acute administration of CAFF increases workload, but has no effects on cardiac contractility in conscious SHHF rats. The cardiac effects are accompanied by increased renin release and NE and E plasma levels. Moreover, this study provides the first evidence that short-term caffeine consumption increases renal renin secretion in heart failure, an effect most likely due to the blockade of intrarenal adenosine receptors. It is possible that long-term activation of neurohumoral mechanisms by CAFF could have adverse effects in heart failure.

Effects of type IV phosphodiesterase inhibition on cardiac function in the presence and absence of catecholamines.

Type IV phosphodiesterase (PDE4) inhibitors may be useful in several diseases in which catecholamine infusions are commonly used, including asthma, sepsis, and multiple organ failure. To determine whether type IV phosphodiesterase inhibitors alter baseline or catecholamine-induced changes in cardiac function or both, we examined the effects of Ro 20-1724 (PDE4 inhibitor) on several cardiac-performance parameters in the absence and presence of norepinephrine, epinephrine, isoproterenol, and dobutamine infusions (3, 1, 0.1, and 3 microg/kg/min, respectively). Male Sprague-Dawley rats received either Ro 20-1724 (10 microg/kg/min; n = 7) or vehicle (n = 6). After a left ventricular catheter was placed and connected to a heart-performance analyzer, each animal received each of the four catecholamines in randomized order (10 min per catecholamine with a 30-min washout period between infusions). In the absence of catecholamines, Ro 20-1724 significantly but mildly (i.e., <10%) increased heart rate but did not alter significantly any other measured cardiac parameter. In addition, Ro 20-1724 did not significantly alter norepinephrine-, epinephrine-, or dobutamine-induced changes in cardiac-performance parameters. There was, however, a significant attenuation of the isoproterenol-induced increase in a single measure of cardiac contractility (maximum dP/dt normalized to pressure). PDE4 inhibition does not cause significant cardiac toxicities in rats, both in the absence and presence of catecholamines. Our data suggest that PDE4 inhibitors may be safely used in critically ill patients receiving catecholamines. A clinical trial of this family of drugs in patients with critical illness is now being planned.

Teaching clinical pharmacology and therapeutics: selective for fourth-year medical students.

Teaching clinical pharmacology remains both a lifelong learning process and a lifelong challenge for clinical pharmacologists and other medical educators. In the current information age, with an explosion of drug-related data, the prime topic for discussion is how to teach clinical pharmacology. This article describes our response to the challenges in developing a selective course in clinical pharmacology, and our experience from the first 2 years of the course. Our emphasis is on how to provide in an efficient way knowledge, skills, and attitudes students will need as physicians in the coming decades. Faculty from the Center for Clinical Pharmacology at the University of Pittsburgh in conjunction with faculty from the University of Pittsburgh School of Medicine have developed a one-month intensive course in clinical pharmacology. The integrated course program consists of four overlapping components: 1) general clinical pharmacology (focused on individualization of drug therapy); 2) rational prescribing principles (general principles of drug selection, how to prepare a personal formulary); 3) disease-specific clinical topics (pharmacotherapy of diseases and medical conditions most commonly seen in routine medical practice); and 4) workshops for special attention topics (pharmacokinetics, pain treatment, toxicology, dialysis). In congruence with established educational goals, the course includes drug-, patient-, and disease-oriented concepts. A variety of learning formats (didactic and interactive lectures, one-day problem-based learning sessions, small group case discussions, self-directed and small group directed learning, quizzes, and computer-assisted learning) are used to teach students how to apply the general concepts of clinical pharmacology and rational pharmacotherapy to clinical medicine, and to prepare them to become independent lifelong learners in therapeutics. Student feedback from the first 2 years of this course indicates that this multi-modal teaching format is effective. The majority of students who took the course in clinical pharmacology in 1997 found it to be very beneficial.

Effects of adenosine deaminase inhibition on blood pressure in old spontaneously hypertensive rats.

Adenosine is an ubiquitously occurring endogenous nucleoside that via cell surface receptors exerts multiple antihypertensive actions, and mediates a number of biological responses that may reduce cardiovascular disease risk. Therefore modulation of endogenous levels of adenosine may offer beneficial effects in hypertension. The objective of this study was to determine whether inhibition of adenosine deaminase lowers blood pressure in spontaneously hypertensive rats (SHR). We investigated the effects of erythro-9-(2-hydroxyl-3-nonyl) adenine (EHNA), an adenosine deaminase inhibitor, on hemodynamic and renal parameters in 16-week-old and 36-week-old SHR and normotensive Wistar Kyoto rats (WKY) and in 36-week-old SHR and WKY pretreated with 1,3-dipropyl-8-p-sulfopheznylxanthine (DPSPX, an adenosine antagonist that does not enter the brain and is restricted to the extracellular space). Adenosine deaminase inhibition with EHNA (10 mg/kg, iv.) produced a marked fall in arterial blood pressure in older (MABP 162.0+/-7.6 mmHg and 120.7+/-11.7 mmHg for baseline and EHNA period, respectively; p<0.01), but not younger, SHR, whereas no effects on blood pressure were observed in age-matched normotensive WKY rats. EHNA did not affect renal hemodynamic and excretory function in any of six groups of animals. DPSPX blocked the antihypertensive effects of EHNA, suggesting that the effects of EHNA on blood pressure are mediated via peripheral adenosine receptors. Further studies are required to elucidate why inhibition of adenosine deaminase lowers blood pressure only in older SHR. The present data suggest that inhibition of adenosine deaminase may provide beneficial effects in older hypertensives and lead us to propose that design and use of extracellular adenosine deaminase inhibitors may offer cardiovascular protection in hypertension.

Central effects of caffeine on renal renin secretion and norepinephrine spillover.

Endogenous adenosine in the brain may inhibit central sympathetic tone and thereby restrain renin release, a mechanism that may be particularly important when sympathetic activity is enhanced. The purpose of our study was to test the hypothesis that the adenosine receptor antagonist caffeine increases renin release in part by disabling the central nervous system (CNS) adenosine brake on renin release. This hypothesis was tested by conducting three protocols in anesthetized rats. In the first protocol, intracerebroventricular (i.c.v.) infusions of caffeine (10 micrograms/kg/min) did not alter either bradycardic responses to intravenous (i.v.) infusion of N6-cyclopentyladenosine (CPA, A1-receptor agonist) or depressor responses to i.v. infusions of CGS21680 (A1-receptor agonist). However, i.c.v. caffeine did block bradycardic responses to i.c.v. boluses of CPA and depressor responses to i.c.v. boluses of CGS21680, thus demonstrating that i.c.v. caffeine at the dose used blocks CNS but not peripheral adenosine receptors. In the second protocol, hydralazine (1 and 10 mg/kg, administered intraperitoneally) significantly enhanced both the renal secretion of renin and the renal spillover of norepinephrine (NE), thus confirming that hydralazine can increase renin release by unloading arterial baroreceptors and increasing sympathetic tone to the kidneys. In the third protocol, the effects of i.c.v. caffeine (10 micrograms/kg/min) on hydralazine-induced (1 and 10 mg/kg, administered intraperitoneally) changes in renal secretion of renin and renal NE spillover were investigated. In this protocol, i.c.v. caffeine did not alter baseline values for either the renal secretion of renin or NE. In contrast, i.c.v. caffeine significantly (p = 0.03) enhanced the increase in renal renin secretion induced by 1 and 10 mg/kg hydralazine (for 1 mg/kg hydralazine delta of 6.4 +/- 46.7 and 142.4 +/- 142.9 renin activity/min/kg body weight in control and caffeine-treated animals, respectively; for 10 mg/kg hydralazine, delta 227.8 +/- 73.9 and 600.8 +/- 168.9 renin activity/min/kg body weight in control and caffeine-treated animals, respectively). The enhanced renin-secretion response to hydralazine in caffeine-treated rats was accompanied by augmented hydralazine-induced increase in renal NE spillover (p = 0.035). These data strongly support the hypothesis of a CNS adenosine brake on renin release that is disabled by caffeine.

Tachyphylaxis to angiotensin II and [Phe4]-angiotensin II in the rat mesentery in vivo.

The existence of tachyphylaxis to angiotensin II (Ang II) in vivo is not unequivocally established, and the hydroxyl group of the Tyr4 residue of Ang II has been suggested as a determinant of tachyphylaxis. In view of these observations, we conducted a series of experiments to assess and compare the ability of Ang II and [Phe4]-Ang II to induce tachyphylaxis in vivo. All experiments were performed in the autoperfused rat mesenteric vascular bed. The tachyphylaxis to intramesenteric (IMA) infusions of Ang II was minimal, and after 120 min of continuous infusion (33 pmol/min), Ang II retained most of its vasoconstrictor activity (67.7 +/- 6.6% of the initial response). Also most of the agonist activity was retained even when Ang II was administered continuously in a dose-increasing manner (1-33 pmol/min). Continuous IMA infusions of single doses of [Phe4]-Ang II caused an initial full response that rapidly declined, and after 120 min retained only 18.2 +/- 9.2% of the initial response. Single continuous IMA infusion of [Phe4]-Ang II induced greater vasoconstrictor responses compared with vascular responses to the same doses delivered after the infusion of smaller doses. This difference was significant (P < .001, unpaired Student's t test) for all doses (10, 100, 330 and 1000 pmol/min) of [Phe4]-Ang II. In conclusion, although the development of tachyphylaxis to Ang II in vivo is minimal, tachyphylaxis to [Phe4]-Ang II does occur (at least in rat mesenteric vascular bed). Thus, omission of the hydroxyl group in position 4 dramatically increases the tachyphylactic potential, while preserving full agonist activity. These results suggest that the hydroxyl group of Tyr4 may protect the Ang II receptor from changes that induce tachyphylaxis.

Possible role of adenosine deaminase in vaso-occlusive diseases.

OBJECTIVE: To describe several emerging concepts regarding the biosynthesis, metabolism and biological roles of adenosine and to illustrate the possible significance of these ideas to vascular biology by proposing a hypothesis pertaining to the role of erythrocyte-derived adenosine deaminase in vaso-occlusive diseases associated with damaged erythrocytes. MULTIPLE PATHWAYS FOR ADENOSINE FORMATION: Three pathways of adenosine biosynthesis have been well established: the intracellular ATP pathway, the extracellular ATP pathway and the transmethylation pathway. A fourth pathway that has received relatively little attention, but could be particularly important in vascular smooth muscle, is the cyclic AMP-adenosine pathway. This pathway involves the extracellular or intracellular metabolism, or both, of cyclic AMP to AMP and hence to adenosine. Our recent experiments in cultured vascular smooth muscle cells, isolated vascular beds and intact animals support the existence of the cyclic AMP-adenosine pathway. Together these four pathways of adenosine formation should assure pharmacologically active levels of vascular adenosine. ANTIVASO-OCCLUSIVE ACTIONS OF ADENOSINE: The overall pharmacologic profile of adenosine suggests that this nucleoside functions to protect vascular beds from vaso-occlusive events. In this regard, some well known effects of adenosine include vasodilation, release of nitric oxide from vascular endothelial cells and inhibition of platelet aggregation, platelet adhesion, neutrophil-endothelial interactions, renin release and noradrenergic neurotransmission. Moreover, studies indicate that adenosine also releases nitric oxide from cultured vascular smooth muscle cells and inhibits vascular smooth muscle cell growth. Thus, any condition that reduces the levels of adenosine in the blood vessel wall or blood vessel-blood interface might predispose toward vaso-occlusive events. DAMAGE TO ERYTHROCYTES REDUCES ADENOSINE LEVELS: Adenosine deaminase rapidly metabolizes adenosine to inosine, which lacks antivaso-occlusive properties. Because erythrocytes are generously endowed with adenosine deaminase, any condition that damages erythrocytes will cause leakage of adenosine deaminase from erythrocytes directly onto the blood vessel wall, thus diminishing local vascular levels of adenosine. Experiments using dimethyl sulfoxide-induced hemolysis have confirmed the hypothesis that erythrocyte-derived adenosine deaminase can reduce adenosine levels in vivo. HYPOTHESIS: THE ROLE OF ERYTHROCYTE-DERIVED ADENOSINE DEAMINASE IN VASO-OCCLUSIVE DISEASES: Because multiple biosynthetic pathways maintain pharmacologically active levels of adenosine within the blood vessel wall and adenosine exerts a number of antivaso-occlusive effects, release of adenosine deaminase from erythrocytes may increase the risk for vaso-occlusive events.

Effects of angiotensin subtype 1 and subtype 2 receptor antagonists in normotensive versus hypertensive rats.

The purpose of this study was to examine in vivo the importance of angiotensin subtype 1 (AT1) versus subtype 2 (AT2) receptors in spontaneously hypertensive (hypertensive) versus normotensive Wistar-Kyoto (control) rats. Intravenous infusions of DuP 753, a selective AT1 receptor antagonist, abolished the pressor responses to intravenous infusions of angiotensin II in both strains, and the potency of DuP 753 in this regard was similar in the two strains. DuP 753 also abolished angiotensin II-induced aldosterone release in both strains; however, with respect to inhibiting angiotensin II-induced aldosterone release, DuP 753 was more potent in hypertensive compared with control rats. In hypertensive but not control rats, DuP 753 inhibited angiotensin II-induced aldosterone release at doses lower than required to inhibit angiotensin II-induced pressor responses. Intramesenteric infusions of DuP 753 abolished mesenteric vascular responses to intramesenteric infusions of angiotensin II with a similar potency in both strains. In control but not hypertensive rats, angiotensin II consistently potentiated noradrenergic neurotransmission in the mesenteric vascular bed, and this effect of angiotensin II was abolished by DuP 753. High doses of PD123177, a selective AT2 antagonist, did not influence any of the aforementioned effects of angiotensin II in either strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Caffeine potentiates vasodilator-induced renin release.

Previous studies strongly suggest that adenosine receptors on juxtaglomerular cells function to restrain the secretion of renin induced by a variety of stimuli. The clinical significance of this is that caffeine, a widely consumed adenosine receptor antagonist, could augment renin release responses to diseases such as renovascular hypertension, liver cirrhosis and heart failure and to therapeutic maneuvers such as salt restriction, diuretics and vasodilators. Caffeine may be particularly troublesome in this regard because this methylxanthine has central nervous system effects and intracellular actions that also might contribute to the overall ability of caffeine to potentiate renin secretion. The purpose of this study was to document the effects of caffeine on renin release responses to a vasodilator and to investigate what mechanisms were responsible for any augmentation of vasodilator-induced renin secretion. Accordingly, we compared the effects of caffeine vs. 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX; a xanthine that we documented in this study not to significantly enter the brain or penetrate cell membranes) on base-line and hydralazine-induced renin release in both normal and beta adrenoceptor-blocked (propranolol, 15 mg/kg) rats. Both xanthines (at a dose of 10 mg/kg plus 150 micrograms/min) attenuated adenosine-mediated hypotension and bradycardia, and DPSPX was at least as effective as caffeine in antagonizing peripheral adenosine receptors. Caffeine and DPSPX increased base-line plasma renin activity to a similar extent regardless of whether the animals were pretreated with propranolol. In rats with an intact beta adrenergic system, caffeine, but not DPSPX, increased the renin release response to low-dose hydralazine (1 mg/kg). Although both xanthines augmented the renin release response to high-dose hydralazine (10 mg/kg), caffeine was more efficacious in this regard. In beta adrenoceptor-blocked rats, neither caffeine nor DPSPX augmented the renin release response to low-dose hydralazine, whereas both xanthines equally potentiated the renin release response to high-dose hydralazine. These data demonstrate that caffeine increases base-line renin release primarily by blocking peripheral (most likely renal), cell-surface adenosine receptors; however, caffeine potentiates vasodilator-induced renin secretion in part by blocking peripheral (most likely renal), cell-surface adenosine receptors and in part by additional central nervous system and/or intracellular mechanism(s) that involve the beta adrenergic system.