Richard E. Klabunde

Dipyridamole inhibition of adenosine metabolism in human blood

The effects of dipyridamole on the metabolism of adenosine were studied in human whole blood. The half-life for adenosine disappearance and the formation of deamination and phosphorylation products were determined by adding [3H]adenosine to blood incubating at 37 degrees C. The initial adenosine concentration was 10 nmol/ml blood. Aliquots of blood were removed at specific times following the addition of labelled adenosine. The aliquots were later assayed for adenosine and its products by reverse-phase high pressure liquid chromatography. The half-life for adenosine in undiluted blood was less than 10 s; therefore, to accurately evaluate the effects of dipyridamole, blood was diluted 1 : 12 in isotonic saline. At this dilution, the half-life for adenosine was 1.3 min. Dipyridamole concentrations of 1 nmol/ml blood caused 90% inhibition of adenosine metabolism. Inhibition was virtually complete, except for plasma deamination, at concentrations greater than 10 nmol/ml blood. Since these inhibitory concentrations of dipyridamole are on the same order as those achieved therapeutically in man, these data indicate that dipyridamole at therapeutic concentrations causes significant inhibition of adenosine metabolism in whole blood.

NG-monomethyl-l-arginine (NMA) restores arterial blood pressure but reduces cardiac output in a canine model of endotoxic shock.

Previous studies have suggested that NMA or similar inhibitors of nitric oxide synthesis from L-arginine reverses or prevents the hypotension associated with endotoxin administration. We wanted to determine if vascular and cardiac responses to NMA support the idea that inhibitors of nitric oxide synthesis might be useful in the treatment of septic shock. Pentobarbital-anesthetized beagle dogs were administered endotoxin for 2 hours at a dose of 250 ng/kg/min. This resulted in reductions in systemic vascular resistance (34% decrease) and mean arterial pressure (25% decrease). Administration of NMA (30 mg/kg, IV) caused large and sustained increases in mean arterial pressure and systemic vascular resistance, and a large decrease in cardiac output and femoral arterial blood flow. Although NMA restored arterial pressure, the large and sustained fall in cardiac output suggests that the cardiovascular action of NMA is detrimental to dogs treated with endotoxin.

Cardiovascular actions of inhibitors of endothelium-derived relaxing factor (nitric oxide) formation/release in anesthetized dogs

NG-monomethyl-L-arginine (NMA) and NG-nitro-L-arginine (NNA), both of which are inhibitors of nitric oxide (endothelium-derived relaxing factor, EDRF) production from L-arginine, have been shown to be pressor agents in vivo. This study compared the cardiac and vascular responses to intraaortic administration of NMA and NNA in anesthetized dogs. NMA at doses of 3, 10, 30 and 100 mg kg-1 i.a. increased systemic vascular resistance and decreased cardiac output; mean arterial pressure increased by 10 mm Hg (at 100 mg kg-1 dose). Heart rate did not change. NNA, administered at doses of 1, 3, 10 and 30 mg kg-1 i.a. produced similar cardiovascular actions and was equipotent to NMA. Pretreatment with indomethacin abolished the pressor response to NMA; however, systemic vasoconstriction and cardiac depression still occurred. Increasing mean arterial pressure by phenylephrine infusion to levels much greater than produced by NMA and NNA caused only small reductions in cardiac output. NMA did not reduce coronary blood flow, but instead caused a transient flow increase. Therefore, systemic administration of NMA and NNA result in pronounced systemic vasoconstriction and cardiac depression with only a small pressor response in anesthetized dogs. The cardiac depression did not result from elevated arterial pressure nor was it due to coronary vasoconstriction and reduced myocardial oxygen supply/demand ratio.

NGMethyl-L-arginine decreases contractility, cGMP and cAMP in isoproterenol-stimulated rat hearts in vitro

NG-Methyl-L-arginine (NMA), an inhibitor of nitric oxide synthesis by vascular endothelium, depresses cardiac function and causes systemic vasoconstriction in vivo. The mechanism of cardiac depression is unclear. Since cGMP inhibits one isoform of myocardial phosphodiesterase (PDE), we hypothesized that a decrease in cGMP might increase PDE activity and lower myocardial cAMP levels, resulting in decreased contractility. Experiments were conducted in isolated, paced, Langendorff-perfused (constant flow) rat hearts under control or isoproterenol-stimulated conditions. In non-stimulated hearts, a 15 min infusion of 30 microM NMA had no effect on cAMP content or on left ventricular dP/dt; however, myocardial cGMP content was decreased. Infusion of 0.01 microM isoproterenol caused dP/dt to increase and caused coronary resistance to fall; myocardial cAMP levels increased while cGMP remained unchanged by isoproterenol. In this stimulated condition, infusion of 30 microM NMA decreased dP/dt and myocardial cGMP and cAMP concentrations. NMA caused coronary resistance to increase to similar maximal values in isoproterenol-stimulated and non-stimulated hearts. Although coronary flow was kept constant during NMA administration, NMA depressed cardiac contractility in isoproterenol-stimulated hearts, but not in non-stimulated hearts, and the depressed contractility in isoproterenol-treated hearts was associated with a decrease in myocardial content of cGMP and cAMP. Therefore, these results are consistent with the hypothesis that NMA may decrease myocardial contractility by decreasing cGMP which leads to increased PDE activity and decreased cAMP.

Nitric Oxide Synthase Inhibition Does Not Prevent Cardiac Depression In Endotoxic Shock

Enhanced production of nitric oxide has been implicated in cardiac and vascular dysfunction associated with septic and endotoxic shock. To test this hypothesis, conscious rats were administered endotoxin. 6 h later, the rats were anesthetized, arterial pressure was measured, and hearts were removed for Langendorff perfusion in the absence and presence of .01 μ M isoproterenol. Left ventricular developed pressure was 61 ± 6 mmHg in control rats and 39 ± 5 mmHg in endotoxin-treated rats. Inotropic responses to isoproterenol were unaffected by endotoxin treatment. Administration of nitric oxide synthase (NOS) inhibitors (NG-nitro-l-arginine and aminoguanidine) prior to endotoxin did not improve left ventricular function in endotoxin-treated rats. Dexamethasone pretreatment, however, prevented endotoxin-induced cardiac depression. These results suggest that cardiac depression during endotoxemia is not caused by NOS activation and increased nitric oxide production. Furthermore, the cardioprotectant actions of dexamethasone are not related to its ability to inhibit inducible NOS expression.

Adenosine metabolism in dog whole blood: effects of dipyridamole.

Abstract The effects of dipyridamole on the metabolism of adenosine added to dog whole blood were studied in vitro at 37°C. The half-lives for 8.8μM and 100μM adenosine were 3.42 and 6.89 min, respectively. Dipyridamole, in concentrations of 10−7 to 10−4M increased the half-life of 8.8μM adenosine 2 to 5-fold. The disappearance rate of adenosine in the presence of 10−4 dipyridamole was similar to the disappearance rate of adenosine in plasma. Inosine formation was enhanced by dipyridamole. Control blood samples not receiving exogenous adenosine showed an increase in endogenous adenosine and inosine during 30 min of incubation. Endogenous production of nucleosides was unaffected by dipyridamole. Analysis showed that endogenous adenosine approached a steady-state concentration of 1μM. The results indicate that the half-life for adenosine in dog whole blood is significantly greater than previously reported and that dipyridamole is effective in inhibiting adenosine disappearance in concentrations as low as 10−7M. The implications of endogenous production of adenosine are discussed.

Diet‐induced obesity and diabetes reduce coronary responses to nitric oxide due to reduced bioavailability in isolated mouse hearts

Aim:  The aim of the present study was to examine nitric oxide (NO)‐mediated coronary vascular responses in a mouse model of obesity and diabetes induced by a high‐fat, high‐carbohydrate diet. We hypothesized that endogenous NO bioavailability would be reduced in obese/diabetic mouse hearts due to enhanced superoxide anion production, and that coronary smooth muscle responses to exogenous NO would be reduced.

Role of Nitric Oxide and Reactive Oxygen Species in Platelet-Activating Factor-Induced Microvascular Leakage

Platelet-activating factor (PAF), released during inflammatory responses, increases microvascular permeability to fluid and macromolecules. Previous studies in the hamster cheek pouch microcirculation have shown that PAF-induced increases in permeability can be diminished by pretreatment with a nitric oxide synthase inhibitor indicating that nitric oxide is required for PAF to cause leakage, although nitric oxide itself does not cause leakage. We evaluated the hypothesis that PAF stimulates the production of reactive oxygen species (ROS) that then react with nitric oxide to form a new species that signals the increase in vascular permeability. The hamster cheek pouch microcirculation was used to quantify the leakage of FITC-dextran following topical application of PAF. PAF-induced leakage was markedly inhibited (70%) by prior superfusion of the cheek pouch with superoxide dismutase and catalase. Superfusing the cheek pouch with ROS generated by xanthine oxidase and hypoxanthine produced leakage similar to that observed with PAF. Pretreating the cheek pouch with a nitric oxide synthase inhibitor (Nω-nitro-L-arginine, L-NA) inhibited ROS-induced leakage by 59% and PAF-induced leakage by 64%. The effects of L-NA and superoxide dismutase plus catalase on PAF-induced leakage were not additive. Systemic administration of mercaptoethylguanidine, a peroxynitrite scavenger, inhibited PAF-induced leakage by 60%. These results suggest that PAF-induced leakage may be mediated by an interaction between ROS and NO, perhaps through the formation of peroxynitrite or one of its products.

Inhibition of endotoxin-induced microvascular leakage by a platelet-activating factor antagonist and 5-lipoxygenase inhibitor.

The sepsis syndrome is associated with increased vascular permeability. Mediators may include platelet-activating factor (PAF) and leukotrienes. We conducted experiments in the hamster cheek pouch to determine the ability of a 5-lipoxygenase (5-LO) inhibitor and a PAF antagonist to attenuate the increase in microvascular permeability that results from tissue exposure to endotoxin. Endotoxin (Escherichia coli serotype 0127:B8) or saline was administered to the surface of the cheek pouch following systemic pretreatment with either the 5-LO inhibitor (Abbott-77523; 20 mg/kg, i.v.) or PAF antagonist (Abbott-84768: 1 mg/kg, i.v.). In control hamsters, endotoxin induced a large increase in fluorescein isothiocyanate-dextran leakage into the cheek pouch interstitium. This leakage was not associated with increased leukocyte rolling or adhesion to venular endothelium. In contrast, groups pretreated with the PAF antagonist or the 5-LO inhibitor showed little or no leakage in response to endotoxin. Therefore, PAF and leukotrienes are important mediators of endotoxin-induced increases in microvascular permeability to macromolecules.

Endotoxin-induced microvascular leakage is prevented by a PAF antagonist and NO synthase inhibitor.

The hamster cheek pouch was used to determine the role of platelet activating factor (PAF) and nitric oxide (NO) in leukocyte adhesion and microvascular leakage of FITC-dextran following systemic administration of endotoxin. Endotoxin (5 mg/kg, i.v.) or vehicle was administered to the hamster 15 min after systemic pretreatment with either a PAF antagonist (Abbott-87648, .1 mg/kg, i.v.) or a nitric oxide synthase inhibitor (L-NAME, 30 mg/kg, i.v.), or 60 min after dexamethasone pretreatment (4 mg/kg, i.p.). Endotoxin alone caused rapid leakage of FITC-dextran from the vascular compartment into the tissue. This leakage was not preceded by either increased leukocyte rolling or adhesion in postcapillary venules. Pretreatment with the PAF antagonist, as well as with dexamethasone, completely blocked endotoxin-induced leakage. L-NAME also blocked endotoxin-induced leakage; however, more than 50% of the hamsters treated with L-NAME died within 2 h after endotoxin administration. These results suggest that PAF and NO are important mediators of microvascular leakage during endotoxemia and that their actions are not dependent on leukocyte adhesion to postcapillary venular endothelium.